Human Melanoblasts in Culture: Expression of BRN2 and Synergistic Regulation by Fibroblast Growth Factor-2, Stem Cell Factor, and Endothelin-3

The Institute for Molecular Bioscience, Center for Functional and Applied Genomics, The University of Queensland, Brisbane, Australia.
Journal of Investigative Dermatology (Impact Factor: 7.22). 12/2003; 121(5). DOI: 10.1046/j.1523-1747.2003.12562.x
Source: OAI

ABSTRACT The BRN2 transcription factor (POU3F2, N-Oct-3) has been implicated in development of the melanocytic lineage and in melanoma. Using a low calcium medium supplemented with stem cell factor, fibroblast growth factor-2, endothelin-3 and cholera toxin, we have established and partially characterised human melanocyte precursor cells, which are unpigmented, contain immature melanosomes and lack L-dihydroxyphenylalanine reactivity. Melanoblast cultures expressed high levels of BRN2 compared to melanocytes, which decreased to a level similar to that of melanocytes when cultured in medium that contained phorbol ester but lacked endothelin-3, stem cell factor and fibroblast growth factor-2. This decrease in BRN2 accompanied a positive L-dihydroxyphenylalanine reaction and induction of melanosome maturation consistent with melanoblast differentiation seen during development. Culture of primary melanocytes in low calcium medium supplemented with stem cell factor, fibroblast growth factor-2 and endothelin-3 caused an increase in BRN2 protein levels with a concomitant change to a melanoblast-like morphology. Synergism between any two of these growth factors was required for BRN2 protein induction, whereas all three factors were required to alter melanocyte morphology and for maximal BRN2 protein expression. These finding implicate BRN2 as an early marker of melanoblasts that may contribute to the hierarchy of melanocytic gene control.

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Available from: Joan Helen Leonard, Sep 28, 2015
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    • "For example, a medium containing stem cell factor (SCF), fibroblast growth factor-2 (FGF2), endothelin-3 (EDN3) and cholera toxin led to a cell phenotype switch into unpigmented precursors, containing immature melanosomes and lacking l-dihydroxyphenylalanine reactivity (tyrosinase activity). These melanoblast cultures expressed high levels of BRN2 (POU3F2, N-Oct-3) an early melanoblast in vitro marker, as compared to melanocytes (Cook et al., 2003). However, one should keep in mind that BRN2 is not expressed in murine melanoblasts in vivo (Colombo et al., 2012). "
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    ABSTRACT: Terminally differentiated cells can be reprogrammed by the transient, ectopic overexpression of different sets of genes into induced pluripotent stem cells (iPSCs). This process not only has considerable implications for regenerative medicine but is also highly relevant to multiple stages of oncogenesis, including melanoma. In other settings, the de-differentiation of normal and tumor cells is also responsible for a phenotype switch which completely changes the cell fate. Conversely, iPSCs as well as embryonic stem cells (ESCs) can be differentiated in vitro toward specific lineages, for example melanocytes, which offer useful models to investigate the genetic and epigenetic mechanisms involved in cellular differentiation. Here, we summarize recent findings regarding the reprogramming and de-differentiation of melanocytic cells as well as the latest differentiation protocols of pluripotent stem cells into the melanocyte lineage.
    European journal of cell biology 11/2013; 93(1-2). DOI:10.1016/j.ejcb.2013.11.006 · 3.83 Impact Factor
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    • "Further improvements to the composition of the culture medium have been reported; however, the cultured MPs continued to produce melanin (4). Cook et al successfully cultured human MPs established from neonatal foreskin in 2003 (5). Based on these studies, we optimized the method for the culture of MPs from hair follicles and performed MP identification analysis. "
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    ABSTRACT: The melanocytes in vitiligo repigmentation are derived predominantly from melanocyte precursors (MPs) present in the outer root sheath (ORS) of hair follicles. The methods currently used for culturing MPs are unstable, and the cultured cells have the capacity to produce melanin. These factors are problematic when conducting in vitro studies to investigate the mechanism of repigmentation. Although 1,25-dihydroxyvitamin D3 (VID) has been demonstrated to be highly effective in the treatment of vitiligo in the clinic, its precise mode of action has yet to be elucidated. In the present study, the method for the culture of MPs from the ORS of hair follicles was optimized and the ability of VID to activate MPs was investigated. The results suggested that the MPs cultured using the optimized method mainly exhibited bipolar morphology. The cells proliferated well and were negative for 3,4-dihydroxy-L-phenylalanine (DOPA) staining. Transmission electron microscopy revealed that the cytoplasm of the MPs contained numerous stage I and stage II melanosomes; however, stage III and IV melanosomes were not observed. Following VID treatment, the MPs showed increased dendritic morphology, the cells stained positive for DOPA and stage III and IV melanosomes appeared in the cells. Western blotting revealed that microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1) and TRP-2 were expressed in the MPs and that VID increased the expression levels of MITF, TYR and TRP-1. However, the levels of MITF, TYR and TRP-1 in the MPs prior to and following VID treatment were significantly lower compared with those in cultured epidermal melanocytes, while the levels of TRP-2 in these three groups were not significantly different. Subsequent to VID treatment, the TYR activity in the MPs increased significantly, as did the corresponding melanin levels. In conclusion, the present study successfully optimized the method for MP culture. The MPs demonstrated no significant TYR activity or melanin synthesis; therefore, the MP cultures exhibited the features of MPs in vivo. In addition, VID significantly promoted the differentiation of MPs.
    Experimental and therapeutic medicine 10/2013; 6(4):967-972. DOI:10.3892/etm.2013.1252 · 1.27 Impact Factor
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    • "Our results revealed that MB-derived cocultures had a higher basal level of DCT expression than MC cocultures, but in response to stimuli showed similar patterns of expression (Figure 1). This is consistent with our previous findings (Cook et al., 2003) of DCT being expressed in MB monocultures and levels being diminished as differentiation to MCs occurred and they became more pigmented. DCT is one of the melanocytic markers of MBs and has been implicated in a delay in eumelanogenesis (Sturm et al., 1995), with its protective cellular role against reactive oxygen species now being realized (Panzella et al., 2011). "
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    ABSTRACT: Variant alleles of the human melanocortin 1 receptor (MC1R) reduce the ability of melanocytes to produce the dark pigment eumelanin, with R alleles being most deficient. Cultured melanocytes of MC1R R/R variant genotype give reduced responses to [Nle(4), D-Phe(7)]α-melanocyte-stimulating hormone (NDP-MSH) ligand stimulation and lower levels of DNA repair than MC1R wild-type strains. p38 controls xeroderma pigmentosum (XP)-C recruitment to DNA damage sites through regulating ubiquitylation of the DNA damage-binding protein 2 (DDB2) protein, and p53 is implicated in the nuclear excision repair process through its regulation of XP-C and DDB2 protein expression. We report the effects of MC1R ligand treatment and UVR exposure on phosphorylation of p38 and p53, and DDB2 protein expression in MC1R variant strains. Wild-type MC1R melanocyte strains grown together with keratinocytes in coculture, when treated with NDP-MSH and exposed to UVR, gave synergistic activation of p38 and p53 phosphorylation, and were not replicated by R/R variant melanocytes, which have lower basal levels of phosphorylated forms of p38. Minor increases in p38 phosphorylation status in R/R variant melanocyte cocultures could be attributed to the keratinocytes alone. We also found that MC1R wild-type strains regulate DDB2 protein levels through p38, but MC1R R/R variant melanocytes do not. This work confirms the important functional role that the MC1R receptor plays in UVR stress-induced DNA repair.
    Journal of Investigative Dermatology 02/2012; 132(5):1452-61. DOI:10.1038/jid.2011.473 · 7.22 Impact Factor
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