NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism

Abstract

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.

Keywords: MITF; UVB; melanosome; nicotinamide nucleotide transhydrogenase; pigmentation; redox regulation.

Figure 1.. Nicotinamide Nucleotide Transhydrogenase (NNT) regulates in vitro pigmentation via a redox-dependent mechanism.

(A) siNNT increases pigmentation. Quantification of intracellular melanin content of UACC257 cells treated with siControl, siNNT, or siTyrosinase for 72 hours (Left Panel) and human primary melanocytes treated with siControl or siNNT for 96 hours (Right Panel); n = 3, analyzed by ordinary one-way ANOVA with Dunnett’s post-test (Left Panel) and unpaired, two-sided t-test (Right Panel). Below the graphs, representative cell pellets of the indicated treatment (1x10⁶ cells). (B) Schema: Pathways of pheomelanin and eumelanin biosynthesis. DHICA, 5,6-dihydroxyindole-2-carboxylic acid; DHI, 5,6-dihydroxyindole. Graphs: UACC257 melanoma cells were treated with siControl, or siNNT for 5 days and eumelanin and pheomelanin were measured using HPLC techniques (n = 3). Absolute pigment levels (Left graph) were analyzed by ordinary two-way ANOVA. The eumelanin/pheomelanin ratio (Right graph) was analyzed by unpaired Student t test. (C) siNNT-induced increased pigmentation of human UACC257 melanoma cells is blocked by NAC (5 mM) or MitoTEMPO (20 μM) (daily treatment for 72 h); n = 3, analyzed by ordinary two-way ANOVA with Šídák’s post-test. (D-E) Quantification of intracellular melanin content of UACC257 cells treated for 72 hours with siControl, siNNT, siIDH1, or siIDH1 + siNNT (D), or with siControl, siNNT, siPGC1 a, or siNNT + siPGC1 a (E); n = 3, analyzed by ordinary one-way ANOVA with Dunnett’s post-test. Below the graphs, representative cell pellets (1 x 10⁶ cells) of the indicated treatments. (F) Overexpression of NNT reduced pigmentation. Melanin content in UACC257 cells that overexpressed NNT (NNT OE) or the corresponding control (Empty Vector) for 12 days; n = 3, analyzed by unpaired, two-sided t-test. All data are expressed as mean ± SEM; *p<0.05, **p<0.01, ****p<0.0001.

Figure 2.. Inhibition of NNT enhances melanosome maturation and tyrosinase protein stability via a redox-dependent mechanism.

(A) Immunoblot analysis of whole cell lysates from UACC257 melanoma cells 72 hours post-treatment with either siControl or siNNT, showing increased tyrosinase, DCT/TRP2, and TYRP1, but not PMEL17 protein levels. Band intensities were quantified by ImageJ, normalized to β-actin, plotted relative to siControl (n = 3), and analyzed by multiple t-tests with the Holm-Šídák post-test. (B-D) siNNT-mediated increased protein stability is blocked by antioxidants. UACC257 cells transfected with siControl or siNNT were treated 24 hours post-transfection with 5 mM NAC (B), 0.1 mM NADPH (C), 20 μM MitoTEMPO (D), or control vehicle for 48 h, followed by CHX treatment. Cells were harvested 0, 1, 2 and 4 h post-CHX treatment for immunoblotting. Band intensities were quantified by ImageJ, normalized to β-actin, and plotted relative to t=0; n = 3, analyzed by repeated measures two-way ANOVA with Šídák’s post-test (Asterisks indicate significance of siControl/vehicle vs. each of the other three groups). (E) Proteasome inhibitor MG132 inhibits tyrosinase protein degradation upon CHX treatment of NNT-overexpressing UACC257 cells. The cells were treated with DMSO or MG132 (10 μM) for 6 h, followed by CHX treatment for 0, 1, 2 and 4 h and immunoblotting. Band intensities were quantified by ImageJ, normalized to β-actin and plotted relative to t = 0; n = 3, analyzed by repeated measures two-way ANOVA with Šídák’s post-test. (F) Enhanced melanosome maturation induced by siNNT in primary human melanocyte cells is blocked by NAC (5 mM) or MitoTEMPO (20 μM) (daily treatment for 96 h). The ratios of late stages (III + IV) to early stages (I + II) are presented. n = 4-5, analyzed by ordinary two-way ANOVA with Šídák’s post-test. (G) Inhibition of melanosome maturation induced by NNT overexpression in primary human melanocytes for 7 days. The ratios of late- to early-stage melanosomes were compared by unpaired, two-sided t-test, n=4 (NNT OE) and n=8 (Empty plasmid). All data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Figure 3.. NNT inhibitors are non-toxic and induce pigmentation of primary melanocytes in vitro and in human skin explants.

(A) Murine melanocytes (Melan-A) showed increased melanin content after incubation with 2 mM 2,3BD or DCC, but not after incubation with palmitoyl-CoA; n =3, analyzed by ordinary one-way ANOVA with Dunnett’s post-test. (B-C) Treatment of primary human melanocytes with different doses of DCC (B, n = 4) or 2,3BD (C, n = 6) for 24 hours yielded decreased GSH/GSSG ratios; analyzed by ordinary one-way ANOVA with Tukey’s (B) or Dunnett’s (C) post-test. (D) A single, one-time topical treatment with 2,3BD (1M or 11M) induces human skin pigmentation after 5 days. Left Panel: Representative images of at least three individual experiments are displayed. Right panel: Reflective colorimetry measurements of skin treated with 2,3BD (higher L* values represent lighter skin tones); n = 3, analyzed by ordinary one-way ANOVA with Dunnett’s post-test. (E) Fontana-Masson staining of melanin in human skin after 2,3BD (50 mM) (i) and hematoxylin & eosin staining (ii) compared with vehicle control (DMSO). (iii) Supranuclear capping in human keratinocytes of 2,3BD- and vehicle control-treated skin displayed by Fontana-Masson staining. (F) NNT inhibitors, 2,3BD or DCC, applied daily at a 50 mM dose resulted in skin darkening after 5 days. Left Pane: Representative images of three individual experiments are displayed. Right panel: Reflective colorimetry measurements of human skin treated with 2,3BD, DCC, or DMSO vehicle (higher L* values represent lighter skin tones;) n = 3, analyzed by ordinary one-way ANOVA with Dunnett’s post-test. (G) Immunofluorescence staining for CPD formation (Red) in human skin treated with 50 mM 2,3BD for 5 consecutive days. On the last day, skin was irradiated with 1000 mJ/cm² UVB. The results show a protective role for 2,3BD from UVB-induced CPD damage. Representative images of three individual experiments are displayed. Scale bar 50μM. Quantified results were normalized to the total number of cells; n = 3, analyzed by ordinary two-way ANOVA with Šídák’s post-test. (H) Measurement of γ-H2AX (Red) in human skin revealed no significant toxicity of 2,3BD, while 2,3-BD-induced pigmentation protected from UVB-induced γ-H2AX formation. Representative images of three individual experiments are displayed Scale bar 50μM. Quantified results were normalized to the total number of cells; n = 3, analyzed by ordinary two-way ANOVA with Šídák’s post-test. All data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Figure 4.. NNT regulates pigmentation in mice, zebrafish and human pigmentation disorders.

(A) Left panel: C57BL/6J mice carrying a 5-exon deletion in the Nnt gene resulting in homozygous loss of NNT activity display increased fur pigmentation compared with C57BL/6NJ wild-type Nnt animals. Right graphs: Mouse fur samples were analyzed for pheomelanin and eumelanin levels by HPLC. n = 3, analyzed by multiple t-tests with the Holm-Šídák post-test. (B) Left panel: Zebrafish overexpressing NNT (NNT OE) display decreased pigmentation in individual melanocytes after 5 days. A representative image has been displayed. Results of mean melanocytic brightness, quantified by pixel-based analysis are shown in the graph at right; Empty plasmid (n = 11 fish; 72 melanocytes), NNT OE (n = 12 fish; 78 melanocytes), analyzed by unpaired, two-sided t-test. (C) Zebrafish with the nnt gene edited using CRISPR/Cas9 (NNT KO) display increased pigmentation after 4 days. A representative image has been displayed. Results of mean melanocytic brightness, quantified by pixel-based analysis are shown in the graph at right; Control (n = 42 fish; 120 melanocytes), NNT KO (n = 50 fish; 96 melanocytes). (D) Zebrafish treated for 24 hours with either 100 μM 2,3BD or 50 μM DCC display increased darkening after 4 days. A representative image has been displayed. Results of mean melanocytic brightness, quantified by pixel-based analysis are shown in the graph at right; DMSO (n = 21 fish; 97 melanocytes), 2,3BD (n = 20 fish; 59 melanocytes), DCC (n = 18 fish; 57 melanocytes), analyzed by ordinary one-way ANOVA with Dunnett’s post-test. (E) Left panel: Human skin specimens from Asian individuals with lentigo or post inflammatory hyperpigmentation were compared to normal skin after staining for NNT, DAPI and Fontana Masson. Representative images of at least 3 samples are displayed (epidermis, E; dermis, D) Graph shows NNT signal intensities normalized to absolute cell numbers (DAPI); n = 3, analyzed by ordinary one-way ANOVA with Dunnett’s post-test. All data are expressed as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5.. Association results for SNPs in the NNT gene with skin color in multiple cohorts.

(A) P-values of SNPs from a meta-analysis of skin color (red) combining association results from 4 worldwide cohorts across 462,885 individuals. For each of the 332 SNPs, its location in the NNT gene is shown in the X axis and the negative logarithm of the P-value is shown in the Y-axis. The SNP with the strongest association, rs574878126, is labeled. The adjusted significance threshold is shown with a dashed line. The NNT gene track and a track of regulatory regions obtained from the Ensembl genome browser are shown below. (B) P-values of SNPs from the UK Biobank for sun protection use (orange) and ease of skin tanning (green). For each SNP, its genomic location is shown in the X axis and negative logarithm of the P-value is shown in the Y-axis. The SNPs with the strongest association for each trait, rs574878126 for sun protection use and rs62367652 for skin tanning, are labeled.

Figure 6.. Association results and properties of SNPs from various human genetic association analyses.

(A-B): Allele frequencies for SNPs in the NNT gene showing most significant associations. (A) Alternative allele frequencies of rs561686035 in various worldwide continental populations, obtained from 1000 Genomes Phase 3. This SNP showed the strongest association in the meta-analysis of skin color and for sun protection use. (B) Alternative allele frequencies of rs62367652 in various worldwide continental populations, obtained from 1000 Genomes Phase 3, are shown. This SNP showed the strongest association for ease of skin tanning (sunburn). (C) Association results for SNPs in the NNT gene with or without conditioning on known pigmentation loci. P-values of SNPs from the Rotterdam Study are shown in this scatterplot. The X-axis represent P-values of SNPs from the standard GWAS analysis of skin pigmentation (not conditioned on any other SNP). P-values from two conditional analyses are plotted on the Y-axis: in blue, P-values conditioning on the three known MC1R SNPs; in orange, P-value conditioning on a larger set of known pigmentation SNPs. A diagonal line in black is shown for reference.