Inhibition of Melanosome Transfer Results in Skin Lightening1

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Inhibition of Melanosome Transfer Results in Skin Lightening1 Miri Seiberg, Christine Paine, Elizabeth Sharlow, Magdalena Eisinger, Stanley S. Shapiro Journal of Investigative Dermatology Volume 115, Issue 2, Pages 162-167 (August 2000) DOI: 10.1046/j.1523-1747.2000.00035.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The effect of RWJ-50353 and SLIGRL on pigmentation in individual melanocytes. Equivalents were treated with SLIGRL (10 μM) and with RWJ-50353 (0.1 μM) for 3 d, followed by F&M staining of histologic sections. Melanocytes were from a Hispanic donor. Images of individual melanocytes are shown. Left panels, untreated control; middle panels, RWJ-50353; right panels, SLIGRL. Scale bar: 10 μM. Journal of Investigative Dermatology 2000 115, 162-167DOI: (10.1046/j.1523-1747.2000.00035.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The PAR-2 pathway affects melanosome ingestion by keratinocytes. Melanosomes were isolated from Melan-A cells according toOrlow et al. (1994). Images of HaCaT keratinocytes untreated (a) or treated with SLIGRL (10 μM, (b)) or RWJ-50353 (10 μM, (c)) for 2 d, followed by a 2h incubation with the isolated melanosomes, extensive wash, and F&M staining. Scale bar: 10 μM. (d) Melanin area per cell, quantified by image analysis. Journal of Investigative Dermatology 2000 115, 162-167DOI: (10.1046/j.1523-1747.2000.00035.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Electron microscopy analysis of the RWJ-50353 effect on equivalents. Representative melanosomes as identified in (a) control and (b) RWJ-50353 (100 μM) treated equivalents. (c) A melanocyte dendrite, containing melanosomes, inside an RWJ-50353-treated keratinocyte. Such structures could not be easily identified in untreated controls. Scale bar: (a, b) 0.1 μM; (c) 0.5 μM. Journal of Investigative Dermatology 2000 115, 162-167DOI: (10.1046/j.1523-1747.2000.00035.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 RWJ-50353-induced depigmentation in vivo. Yucatan swine were treated with vehicle (a), and 10 μM (b), 50 μM (c), and 250 μM (d) of RWJ-50353 for 8 wk. (a) Picture of the swine (both sides) after 8 wk of treatment. (b) Chromameter measurements of skin color (L* scale, 0=black, 100=white) during the treatment phase. Both a dose response and a time response are observed. Journal of Investigative Dermatology 2000 115, 162-167DOI: (10.1046/j.1523-1747.2000.00035.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Histologic analysis of swine skin samples treated with RWJ-50353. Swine were treated as described in Figure 4 and biopsies were taken at the eighth week of treatment. F&M staining of skin sections revealed a dose-dependent reduction in melanin deposition at treated sites: (a) vehicle; (b)–(d) 10 μM, 50 μM, and 250 μM of RWJ-50353. Scale bar:20 μM. (e) Relative pigmentation, calculated as melanin area per epidermis area and normalized to untreated controls, obtained by image analysis of F&M-stained skin sections. Journal of Investigative Dermatology 2000 115, 162-167DOI: (10.1046/j.1523-1747.2000.00035.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 The depigmenting effect of RWJ-50353 is reversible. Yucatan swine were treated for 8 wk with 250 μM of RWJ-50353, and followed without treatment for a further 4 wk. Biopsies were taken before the start of treatment (a), after 8 wk (completion of treatment phase, b), and at the ninth, tenth, and twelfth weeks (c–e, 1–4 wk after treatment was terminated). Scale bar: 12 μM. F&M-stained sections revealed re-pigmentation after treatment had been stopped, with no irritation or other side-effects. Differences in epidermal thickness result from the different sites of biopsies, and have no correlation to treatments. (f) Image analysis of F&M-stained sections was used to quantify pigmentation, relative to the untreated control. Journal of Investigative Dermatology 2000 115, 162-167DOI: (10.1046/j.1523-1747.2000.00035.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Electron micrpscopy analysis of Yucatan swine skin treated with RWJ-50353. (a) A representative melanosome inside a keratinocyte of untreated swine skin. (b), (c) Representative melanosomes inside keratinocytes of RWJ-50353 (250 μM) treated swine skin are smaller and less pigmented than control ones. (d) A random distribution of melanosomes in control swine epidermis. (e) Melanosomes of RWJ-50353 (250 μM) treated swine skin are detected mainly at the epidermal–dermal border (marked). Scale bar: (a-c) 0.05 μM; (d, e) 0.8 μM. Journal of Investigative Dermatology 2000 115, 162-167DOI: (10.1046/j.1523-1747.2000.00035.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions