Differentiation of Murine Melanocyte Precursors Induced by 1,25-Dihydroxyvitamin D3 Is Associated with the Stimulation of Endothelin B Receptor Expression

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Differentiation of Murine Melanocyte Precursors Induced by 1,25-Dihydroxyvitamin D3 Is Associated with the Stimulation of Endothelin B Receptor Expression Hidenori Watabe, Yoshinao Soma, Yoko Kawa, Masaru Ito, Shiho Ooka, Kayoko Ohsumi, Takako Baba, Tamihiro Kawakami, Eri Hosaka, Satoko Kimura, Masako Mizoguchi Journal of Investigative Dermatology Volume 119, Issue 3, Pages 583-589 (September 2002) DOI: 10.1046/j.1523-1747.2002.00116.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 1,25(OH)2D3 inhibits cell proliferation in a concentration-dependent manner. NCC-melb4 cells were plated at 2000 cells per well in 96-well plates and were incubated with various concentrations of 1,25(OH)2D3. After 5 d, cells were incubated with alamar blue dye and the fluorescence was measured on a microplate reader. **p < 0.01. Journal of Investigative Dermatology 2002 119, 583-589DOI: (10.1046/j.1523-1747.2002.00116.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Effects of 1,25(OH)2D3 on tyrosinase expression, DOPA reaction and EDNRB expression. NCC-melb4 cells before (a,c,e) and after (b,d,f) treatment with 10-7 M 1,25(OH)2D3 for 72 h were immunohistochemically stained for tyrosinase (a,b) or EDNRB (e,f), or were tested for DOPA reaction (c,d). Scale bar = 10 µm. Journal of Investigative Dermatology 2002 119, 583-589DOI: (10.1046/j.1523-1747.2002.00116.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Enhanced expression of tyrosinase after 1,25(OH)2D3 treatment. Tyrosinase expression in NCC-melb4 cells before and after the treatment with 10-7 M 1,25(OH)2D3 for 72 h was determined by western blotting (a) and by reverse transcription–PCR analysis (b) as described in Materials and Methods. (b)Lanes 1 and 2: β-actin; lanes 3 and 4: tyrosinase. Journal of Investigative Dermatology 2002 119, 583-589DOI: (10.1046/j.1523-1747.2002.00116.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 EDNRB expression is induced by 1,25(OH)2D3 in NCC-melb4 cells but not in NCC-melan5 cells.(a) NCC-melb4 cells were incubated with or without 10-7 M 1,25(OH)2D3 for 72 h. Total RNA was extracted and analyzed by reverse transcription–PCR. Lanes 1 and 2: β-actin; lanes 3 and 4: EDNRB. (b) NCC-melb4 cells were incubated with various concentrations of 1,25(OH)2D3 for 72 h. Total RNA was extracted and analyzed by reverse transcription–PCR. Lane 1: 10-7 M; lane 2: 10-8 M; lane 3: 10-9 M; lane 4: size marker. (c) NCC-melan5 cells were incubated with or without 10-7 M 1,25(OH)2D3 for 72 h. Total RNA was extracted and analyzed by reverse transcription–PCR. Lanes 1 and 2: β-actin; lanes 3 and 4: EDNRB. Journal of Investigative Dermatology 2002 119, 583-589DOI: (10.1046/j.1523-1747.2002.00116.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Reverse transcription–PCR analysis of VDR mRNA expression in NCC-melb4 cells and in NCC-melan5 cells. Total RNA was reverse-transcribed followed by 40 cycles PCR of cDNA using the primers described in Materials and Methods. Lane 1: β-actin of NCC-melb4 cells; lane 2: β-actin of NCC-melan5 cells; lane 3: VDR of NCC-melb4 cells; lane 4: VDR of NCC-melan5 cells. Journal of Investigative Dermatology 2002 119, 583-589DOI: (10.1046/j.1523-1747.2002.00116.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Electron microscopy and ultrastructural DOPA staining reveal the development of melanocyte precursors with 1,25(OH)2D3. Electron microscopy (a,c) and electron microscopic DOPA staining (b,d) of NCC-melb4 cells were made before (a,b) and after (c,d) 72 h of treatment with 10-7 M 1,25(OH)2D3. Before the treatment, NCC-melb4 cells contained only stage I melanosomes (small arrow) and were DOPA negative. After the treatment, stage III–IV melanosomes (large arrows) were seen, and both Golgi areas and melanosomes were DOPA-positive. Scale bar = 1 µm. Journal of Investigative Dermatology 2002 119, 583-589DOI: (10.1046/j.1523-1747.2002.00116.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Induction of MITF mRNA by combined treatment with 1,25(OH)2D3 and EDN3. Reverse transcription–PCR analysis of MITF mRNA from NCC-melb4 cells treated with or without 10-7 M 1,25(OH)2D3 and 100 nM EDN3 for 72 h. Lane 1: no addition; lane 2: 1,25(OH)2D3; lane 3: 1,25(OH)2D3 + EDN3; lane 4: size marker. Journal of Investigative Dermatology 2002 119, 583-589DOI: (10.1046/j.1523-1747.2002.00116.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 1,25(OH)2D3 stimulates the expression of EDNRB in primary NCC cultures. Neural tubes of C57BL/6 mice were explanted and cultured with or without 10-7 M 1,25(OH)2D3. The explants were fixed at 13 d in culture and were subjected to immunohistochemical staining for EDNRB. Note that numerous EDNRB-positive cells (reddish staining) are seen in 1,25(OH)2D3-treated cells (b), but not in nontreated cells (a). Journal of Investigative Dermatology 2002 119, 583-589DOI: (10.1046/j.1523-1747.2002.00116.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 1,25(OH)2D3 increases DOPA-positive cells in primary NCC cultures. Neural tubes of C57BL/6 mice were explanted and cultured with or without 10-7 M 1,25(OH)2D3. The explants were fixed at 13 d in culture and were subjected to the DOPA reaction. Note that numerous DOPA-positive cells are seen in 1,25(OH)2D3-treated cells (b), but not in nontreated cells (a). Journal of Investigative Dermatology 2002 119, 583-589DOI: (10.1046/j.1523-1747.2002.00116.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions