UCHL1 Regulates Melanogenesis through Controlling MITF Stability in Human Melanocytes  Eun Young Seo, Seon-Pil Jin, Kyung-Cheol Sohn, Chi-Hyun Park, Dong Hun Lee, Jin Ho Chung  Journal of Investigative Dermatology  Volume 137, Issue 8, Pages 1757-1765 (August 2017) DOI: 10.1016/j.jid.2017.03.024 Copyright © 2017 The Authors Terms and Conditions

Figure 1 UCHL1 inhibits melanin synthesis in normal human primary epidermal melanocytes (NHEMs) and MNT-1 cells. (a) Immunoblot analysis of UCHL1 expression levels in NHEMs, MNT-1 cells, or normal human epidermal keratinocytes (NHEKs). (b) Both NHEMs and MNT-1 cells were infected with an adenovirus expressing GFP or GFP-UCHL1 at a multiplicity of infection of 30 for 1 hour, and were further incubated for 3 days. Cell pellets were imaged. Scale bar = 5 mm. (c) Melanin contents were measured, with synthetic melanin used as a standard control, and normalized to total protein concentration. (d) NHEMs were transfected with UCHL1 siRNA 100 nM for overnight and incubated for a total of 6 days. Melanin contents were measured as described in the Materials and Methods section. Values represent the mean ± SEM of data from five separate experiments. *P < 0.05, ***P < 0.001. GFP, green fluorescent protein; MNT-1, human melanoma cells; SEM, standard error of the mean; siRNA, small interfering RNA; UCHL1, ubiquitin carboxyl-terminal hydrolase L1. Journal of Investigative Dermatology 2017 137, 1757-1765DOI: (10.1016/j.jid.2017.03.024) Copyright © 2017 The Authors Terms and Conditions

Figure 2 UCHL1 regulates tyrosinase (TYR), dopachrome tautomerase (DCT), and tyrosinase-related protein-1 (TRP-1) protein levels. (a) NHEMs were infected with adenovirus (Ad)-GFP or Ad-GFP-UCHL1 (MOI = 30) for 1 hour, and further incubated for 3 days. Cells were collected for the analysis of protein expression levels. Tyrosinase, TRP-1, and DCT proteins were detected and quantified by western blot analysis. (b) NHEMs were transfected with 100 nM of UCHL1 siRNA for overnight, and incubated for a total of 6 days. Cells were collected for the analysis of protein expression levels. TYR, TRP-1, and DCT proteins were detected and quantified by western blot analysis. Actin was used as a loading control. Data shown are representative of five and six independent experiments, respectively. *P < 0.05, ***P < 0.001. GFP, green fluorescent protein; MOI, multiplicity of infection; NHEM, normal human primary epidermal melanocyte; siRNA, small interfering RNA; UCHL1, ubiquitin carboxyl-terminal hydrolase L1. Journal of Investigative Dermatology 2017 137, 1757-1765DOI: (10.1016/j.jid.2017.03.024) Copyright © 2017 The Authors Terms and Conditions

Figure 3 UCHL1 downregulates MITF levels through negative regulation of protein stability. (a) Immunoblot analysis and quantification of MITF expression in NHEMs expressing adenovirus (Ad)-GFP-UCHL1 (MOI = 30) for 3 days (left panel), or transfected with UCHL1 siRNA 100 nM for 6 days (right panel). (b) Immunoblotting of MITF expression in NHEMs and MNT-1 cells treated with Ad-GFP-UCHL1 at various MOI for 3 days. (c) NHEMs were infected with Ad-GFP or Ad-GFP-UCHL1 (30 MOI), and after 60 hours, the cells were treated with 20 μg/ml cycloheximide (CHX). The cells were harvested at the indicated time points to measure MITF protein levels. To quantify the MITF protein levels, actin was used for normalization and then normalized to the t = 0.5 hours. Values represent from three independent experiments. Statistical analyses were performed using two-way ANOVA. *P < 0.05, ***P < 0.001. ANOVA, analysis of variance; GFP, green fluorescent protein; MITF, microphthalmia-associated transcription factor; MNT-1, human melanoma cells; MOI, multiplicity of infection; NHEM, normal human primary epidermal melanocyte; siRNA, small interfering RNA; UCHL1, ubiquitin carboxyl-terminal hydrolase L1. Journal of Investigative Dermatology 2017 137, 1757-1765DOI: (10.1016/j.jid.2017.03.024) Copyright © 2017 The Authors Terms and Conditions

Figure 4 UCHL1 regulates MITF through its enzyme activity and increases the ubiquitinated form of MITF protein. (a) UCHL1-dependent MITF protein degradation was recovered in the presence of MG132. NHEMs were infected with adenovirus (Ad)-GFP or Ad-GFP-UCHL1 (MOI = 30) for 3 days, and treated with MG132 (150 nM) for 3 hours before cell harvest. (b) UCHL1 interacted with ubiquitinated (Ub)-MITF. NHEMs were infected with Ad-GFP-UCHL1 for 3 days. After 24 hours, NHEMs were transfected with HA-Ub (10 μg), and at day 3, were treated with MG132 (150 nM) for 3 hours before cell harvest. Cell lysates were split into two parts, and each portion was immunoprecipitated with an anti-GFP antibody or IgG as a control, and then detected using an anti-MITF antibody. (c) MNT-1 cells were coinfected with Ad-FLAG-MITF and GFP or Ad-GFP-UCHL1 (MOI 60). Cell lysates were immunoprecipitated with an anti-FLAG antibody and then detected using an anti-HA antibody. (d) MNT-1 cells were transfected with one of the following vectors: myc empty, wild-type UCHL1, S18Y, or C90S mutant UCHL1. GFP, green fluorescent protein; HA-Ub, hemagglutinin-ubiquitin; IB, immunoblotting; IP, immunoprecipitation; MITF, microphthalmia-associated transcription factor; MNT-1, human melanoma cells; MOI, multiplicity of infection; NHEM, normal human primary epidermal melanocyte; UCHL1, ubiquitin carboxyl-terminal hydrolase L1; WT, wild type. Journal of Investigative Dermatology 2017 137, 1757-1765DOI: (10.1016/j.jid.2017.03.024) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Overexpression of MITF restored the UCHL1-induced reduction in melanogenesis. MNT-1 cells were coinfected with an adenovirus containing GFP or GFP-UCHL1 and LacZ or MITF (GFP and LacZ were used as controls). (a) The expression of MITF was examined by western blot analysis. Actin was used as a protein loading control. The results from four independent experiments were quantified using image J software. (b) At day 3, the cells were washed with phosphate-buffered saline once, and melanin levels were quantified by measuring absorbance at 405 nm. Values represent the mean ± SEM of data from three independent experiments. *P < 0.05, **P < 0.01. NS, nonsignificant; GFP, green fluorescent protein; MITF, microphthalmia-associated transcription factor; SEM, standard error of the mean; UCHL1, ubiquitin carboxyl-terminal hydrolase L1. Journal of Investigative Dermatology 2017 137, 1757-1765DOI: (10.1016/j.jid.2017.03.024) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Schematic diagram showing the role of UCHL1 in melanogenesis. UCHL1 inhibited the expression of MITF via activation of ubiquitin-proteasome pathway-mediated degradation, thus leading to decreased melanogenesis in human melanocytes. MITF, microphthalmia-associated transcription factor; UCHL1, ubiquitin carboxyl-terminal hydrolase L1. Journal of Investigative Dermatology 2017 137, 1757-1765DOI: (10.1016/j.jid.2017.03.024) Copyright © 2017 The Authors Terms and Conditions