Variation in Hsp70-1A Expression Contributes to Skin Color Diversity Daiki Murase, Akira Hachiya, Rachel Fullenkamp, Anita Beck, Shigeru Moriwaki, Tadashi Hase, Yoshinori Takema, Prashiela Manga  Journal of Investigative Dermatology  Volume 136, Issue 8, Pages 1681-1691 (August 2016) DOI: 10.1016/j.jid.2016.03.038 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Differences in protein expression in human melanocytes with varying pigmentation levels. (a) NHEMs were cultured and proteins were harvested (three AA- and three white-derived). Two-DGE was performed followed by silver staining. Indicated spots were extracted and subjected to mass spectrometry. (b) Expression of Hsp70-1A, tyrosinase, or TYRP1 in NHEMs was determined by western-blot analysis and (c) densitometry (relative intensity of each band normalized to α-tubulin is expressed to compare protein expression in AA and white melanocytes). Left: Hsp70-1A, center: tyrosinase, right: TYRP1. Values represent means (± SD), n = 5 (different melanocyte lines and/or group); **P < 0.01; *P < 0.05. (d) Correlations are shown between Hsp70-1A and tyrosinase (left) or TYRP1 (right) protein expression levels normalized to α-tubulin expression. AA, African American; DGE, dimensional gel electrophoresis; Hsp70-1A, heat shock protein 70-1A; NHEMs, normal human epidermal melanocytes; TYRP1, tyrosinase-related protein 1. Journal of Investigative Dermatology 2016 136, 1681-1691DOI: (10.1016/j.jid.2016.03.038) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Differential Hsp70-1A expression and colocalization with tyrosinase in AA and white NHEMs and skin. (a) Epidermal proteins, harvested from skin obtained from AA or white donors, were subjected to western blotting for Hsp70-1A expression. β-Actin = loading control. Immunohistologic analysis of (b) Hsp70-1A (red) and tyrosinase (green) or (c) Hsp70-1A (red) and TYRP1 (green) in AA- or white-derived skin was performed. Merged images of Hsp70-1A, tyrosinase or TYRP1, and DAPI nuclear staining are also shown. Lower insets show the magnified areas of the white rectangle-surrounding areas. Bar = 100 μm. (d) AA- or white-derived NHEMs were stained with Hsp70-1A- (red), tyrosinase- (green) and calnexin- (cyan) specific antibodies. Merged images with DAPI are also shown. Lower-right insets show magnified areas. Bar = 20 μm. AA, African American; DAPI, 4',6-diamidino-2-phenylindole; Hsp70-1A, heat shock protein 70-1A; NHEMs, normal human epidermal melanocytes; TYRP1, tyrosinase-related protein 1. Journal of Investigative Dermatology 2016 136, 1681-1691DOI: (10.1016/j.jid.2016.03.038) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Hsp70-1A expression correlates with melanogenesis. (a) AA- or white-derived NHEMs were treated with Hsp70-inhibitor VER-155008 (at increasing concentrations) for 7 days and melanin content determined (mean ± SD, n = 3; **P < 0.01, *P < 0.05 (ANOVA, Dunnett). (b) NHEMs were incubated with 3 μM VER-155008 (for increasing times) and tyrosinase expression determined by western blotting. Densitometry values normalized to β-actin are shown. (c) NHEMs were transfected with HSPA1A-specific or nonspecific siRNAs and melanin quantitated after 7 days. (d) NHEMs were transfected with HSPA1A-specific or nonspecific siRNAs for 3 days and Hsp70-1A, tyrosinase, TYRP1, or TYRP2 expression determined by western blotting. White skin-derived NHEMs were treated with Hsp70-activator SW-02 for (e) 7 days and melanin quantitated or (f) 2 days for western-blot analysis of tyrosinase, TYRP1, TYRP2, or Rab27a expression. β-Actin = loading control. AA, African American; Hsp70-1A, heat shock protein 70-1A; NHEMs, normal human epidermal melanocytes; siRNAs, small interfering RNAs; TYRP, tyrosinase-related protein. Journal of Investigative Dermatology 2016 136, 1681-1691DOI: (10.1016/j.jid.2016.03.038) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Hsp70-1A inhibition significantly lightens skin color in three-dimensional human skin substitutes. AA- or white-derived NHEMs transfected with two different HSPA1A- or nonspecific siRNAs were cultured with AA-derived NHEKs for 20 days to generate three-dimensional human skin substitutes (3D-HSSs). (a) Representative images of 3D-HSSs including AA-derived NHEMs are shown. 3D-HSSs were subjected to (b) Fontana-Masson staining (scale bar = 100 μm) or (c) harvested and solubilized to quantitate melanin content. 3D-HSSs were cultured for 15 days and proteins were harvested. (d) Western-blot analysis and (e) densitometry were performed to determine tyrosinase and TYRP2 expression (β-actin = loading control). Graphs show relative expression after normalization against β-actin (left: tyrosinase, right: TYRP2). Values represent mean ± SD from three different samples. **P < 0.01; *P < 0.05 (t-test). AA, African American; Hsp70-1A, heat shock protein 70-1A; NHEMs, normal human epidermal melanocytes; siRNAs, small interfering RNAs; TYRP2, tyrosinase-related protein 2. Journal of Investigative Dermatology 2016 136, 1681-1691DOI: (10.1016/j.jid.2016.03.038) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Hsp70-1A activity correlates with pigmentation of cultured skin. Skin from an AA donor was cultured with or without VER-155008 for 8 days (indicated concentrations). (a) Photographs show representative samples (scale bar = 2 mm). (b) Treated skin was subjected to Fontana-Masson staining. Scale bars = 100 μm. (c) Colorimeter measurements of pigmentation were also taken (L* values, means ± SD, n = 3; **P < 0.01 [ANOVA, Dunnett]). (d) Proteins were harvested and subjected to western-blot analysis of TYRP1 expression. β-Actin = loading control. Skin from a white donor was cultured with or without SW-02 for 8 days (indicated concentrations). (e) Photographs of representative samples are shown (scale bars = 2 mm). (f) Fontana-Masson staining of SW-02-treated skin was performed. Scale bars = 100 μm. AA, African American; Hsp70-1A, heat shock protein 70-1A; TYRP1, tyrosinase-related protein 1. Journal of Investigative Dermatology 2016 136, 1681-1691DOI: (10.1016/j.jid.2016.03.038) Copyright © 2016 The Authors Terms and Conditions

Figure 6 Hsp70-1A expression inversely correlates with autophagy-mediated melanosome degradation. (a) Hsp70-1A expression in four AA and four white skin-derived NHEKs was determined by western blotting and (b) densitometry (mean ± SD, ***P < 0.001 [t-test]). (c) AA NHEKs were transfected with HSPA1A-targeting or nonspecific siRNAs with or without HCQ. LC3 expression was determined by western blotting and (d) densitometry (mean ± SD, *P < 0.05 [t-test]). (e) AA NHEKs were cultured with or without VER-155008 (3 μM). LC3-, p62-, phospho-p70S6K, and p70S6K expression was determined by western-blot analysis and (f) densitometry (**P < 0.01 [ANOVA, Holm test]). NHEKs were cultured with isolated melanosomes for 24 hours, washed, and then cultured with or without (g) VER-155008 (AA NHEKs) or (h) SW-02 (white NHEKs) for 48 hours. Pmel17, LC3, and p62 expression was determined by western blotting. β-Actin/α-tubulin = loading control. AA, African American; HCQ, hydroxychloroquine; Hsp70-1A, heat shock protein 70-1A; NHEMs, normal human epidermal melanocytes; siRNAs, small interfering RNAs. Journal of Investigative Dermatology 2016 136, 1681-1691DOI: (10.1016/j.jid.2016.03.038) Copyright © 2016 The Authors Terms and Conditions