IFN-γ signaling maintains skin pigmentation homeostasis through regulation of melanosome maturation

Abstract

Cellular homeostasis is an outcome of complex interacting processes with nonlinear feedbacks that can span distinct spatial and temporal dimensions. Skin tanning is one such dynamic response that maintains genome integrity of epidermal cells. Although pathways underlying hyperpigmentation cascade are recognized, negative feedback regulatory loops that can dampen the activated melanogenesis process are not completely understood. In this study, we delineate a regulatory role of IFN-γ in skin pigmentation biology. We show that IFN-γ signaling impedes maturation of the key organelle melanosome by concerted regulation of several pigmentation genes. Withdrawal of IFN-γ signal spontaneously restores normal cellular programming. This effect in melanocytes is mediated by IFN regulatory factor-1 and is not dependent on the central regulator microphthalmia-associated transcription factor. Chronic IFN-γ signaling shows a clear hypopigmentation phenotype in both mouse and human skin. Interestingly, IFN-γ KO mice display a delayed recovery response to restore basal state of epidermal pigmentation after UV-induced tanning. Together, our studies delineate a new spatiotemporal role of the IFN-γ signaling network in skin pigmentation homeostasis, which could have implications in various cutaneous depigmentary and malignant disorders.

Keywords: detanning; gene regulation; interferon; melanin.

Fig. 1.

Analysis of the pigmentation oscillator reveals a dominant IFN-γ signature in depigmented cells. (A) Schematic representation of the in vitro biological oscillator showing two cycles of pigmentation and depigmentation of cultured cells at different densities (arrows indicate the time of plating). (Upper) Cell pellets of B16 cells on different days for cycle 1. (B, Left) Time-dependent changes in the relative expression of all genes in the oscillator model. (B, Right) Normalized coefficient value obtained by FT analysis of all genes as a function of their respective frequencies. (C, Left) Relative expression level of known pigmentation genes through two cycles of pigmentation and depigmentation. (C, Right) Relative expression level of known IFN-γ–regulated genes identified by FT analysis across the two cycles. (D) Cell pellets of control (C) and IFN-γ–treated B16 cells at day 12.

Fig. 2.

IFN-γ signaling mediates hypopigmentation of primary human melanocytes by arresting melanosome maturation. (A, Upper) Cell pellets of control and IFN-γ–treated primary human epidermal melanocytes after 7 d of treatment. (A, Lower) Fold change in gene expression levels of IFN-γ–treated melanocyte cultures expressed as mean fold change ± SEM across three independent cultures. (B) Western blot analysis of DCT, TYR, and MITF proteins after IFN-γ treatment as a function of time. C, control. (C) TEM images of (Left) control and (Right) IFN-γ–treated melanocytes. (D) Corresponding confocal images of melanocytes treated with IFN-γ stained with HMB45 (green). (Scale bar: C, 2 μm; D, 10 μm.)

Fig. 3.

IFN-γ signaling mediates reversible hypopigmentation of melanocytes independent of MITF. (A) Experimental setup for studying reversibility of IFN-γ effect on melanocytes. Cell pellet of primary human melanocytes treated with IFN-γ for 7 d; the cells were allowed to recover for 5 d (day 12) by the removal of IFN-γ. Western blot analysis of DCT, TYR, and MITF proteins of untreated and IFN-γ–treated cells (day 7) and day 12 untreated and recovered cells. C, control; I, IFN. (B) Cell pellets of melanocytes. The levels of proteins by Western blot analysis and Tyr activity determined by an in-gel L-dopa assay of cells treated with 600 nM α-MSH and 100 μM isobutyl methyl xanthine (IBMX) for 5 d in the presence and absence of IFN-γ are shown. (C) Dct promoter (3.7 kb) activity in the presence of 600 nM α-MSH and 100 U/mL IFN-γ (alone or combined) as measured by dual luciferase assay. RLU, relative light units. (D) Promoter activity of a 3.7-kb WT Dct promoter and M-Box–deleted construct in the presence of α-MSH and IFN-γ. ***P < 0.001; ns, not significant.

Fig. 4.

Mechanism of Irf1-mediated down-regulation of Dct. (A) Levels of DCT mRNA as measured by real-time PCR in mock vs. JAK-1, STAT-1, IRF1, IRF3, IRF7, and IRF9 siRNA-transfected melanocytes in the absence or presence of IFN-γ. (B) mRNA levels of transcripts in cells transfected with Irf1 expression construct compared with mock-transfected B16 cells. UT, untransfected. (C) Binding of Irf1 to biotinylated Dct promoter in avidin pull down from IFN-γ–treated B16 cell lysates was detected by Western blot (WB). (D) Dct promoter activity in the absence and presence of varying concentrations of Irf1 expression plasmid (Dct-luciferase: Irf1 plasmid DNA, 1:0, 1:0.5, and 1:1). Bars represent mean ± SD across replicates. ***P < 0.001. (E) Dual luciferase reporter assay carried out with several deletion constructs of Dct promoter in the presence and absence of IFN-γ. Bars represent mean ± SD across replicates.

Fig. 5.

Physiological and pathophysiological roles of IFN-γ in skin pigmentation. (A) Images of IFNG^+/+ and IFNG^−/− mice in C57BL/6 background at 6 wk. Arrows indicate pigmentation at the sites where epidermal melanocytes are present. (B) Western blot analysis of Dct and Tyr proteins from the tail epidermis in IFNG^+/+ and IFNG^−/− mice. (C) Image analysis for quantitating levels of pigmentation. Bar graphs indicate average ± SEM across 22 measurements across the two ear lobes. **P < 0.005; ***P < 0.001. (D) Comparative real-time PCR analysis for a panel of known IFN-γ–modulated genes in nonlesional vs. lesional epidermis of patients with different manifestations of leprosy (Lep). Dashed line represents a cutoff of twofold considered as significant for up-regulation.