UV exposure modulates hemidesmosome plasticity, contributing to long-term pigmentation in human skin

Abstract

Human skin colour, ie pigmentation, differs widely among individuals, as do their responses to various types of ultraviolet radiation (UV) and their risks of skin cancer. In some individuals, UV-induced pigmentation persists for months to years in a phenomenon termed long-lasting pigmentation (LLP). It is unclear whether LLP is an indicator of potential risk for skin cancer. LLP seems to have similar features to other forms of hyperpigmentation, eg solar lentigines or age spots, which are clinical markers of photodamage and risk factors for precancerous lesions. To investigate what UV-induced molecular changes may persist in individuals with LLP, clinical specimens from non-sunburn-inducing repeated UV exposures (UVA, UVB or UVA + UVB) at 4 months post-exposure (short-term LLP) were evaluated by microarray analysis and dataset mining. Validated targets were further evaluated in clinical specimens from six healthy individuals (three LLP+ and three LLP-) followed for more than 9 months (long-term LLP) who initially received a single sunburn-inducing UVA + UVB exposure. The results support a UV-induced hyperpigmentation model in which basal keratinocytes have an impaired ability to remove melanin that leads to a compensatory mechanism by neighbouring keratinocytes with increased proliferative capacity to maintain skin homeostasis. The attenuated expression of SOX7 and other hemidesmosomal components (integrin α6β4 and plectin) leads to increased melanosome uptake by keratinocytes and points to a spatial regulation within the epidermis. The reduced density of hemidesmosomes provides supporting evidence for plasticity at the epidermal-dermal junction. Altered hemidesmosome plasticity, and the sustained nature of LLP, may be mediated by the role of SOX7 in basal keratinocytes. The long-term sustained subtle changes detected are modest, but sufficient to create dramatic visual differences in skin colour. These results suggest that the hyperpigmentation phenomenon leading to increased interdigitation develops in order to maintain normal skin homeostasis in individuals with LLP.

Keywords: hemidesmosome; pigmentation; skin; sunburn; ultraviolet radiation.

Figure 1

Long-term LLP hyperpigmentation increases levels of melanin in the basal layer of human skin. (A) Images of skin after a single erythemal UV treatment at > 9 months post-exposure are depicted for the 3 LLP+ and 3 LLP- subjects from which biopsy specimens used for immunohistochemistry were acquired from the most pigmented UV-exposed area versus the adjacent unexposed control (insets show pigmentation in the UV-treated areas after 2 weeks). (B) Melanin contents of specimens detected by Fontana-Masson staining; representative micrographs are shown for #S100. scale bar = 50 μm. (C) Melanin content (% Positive Area) and distribution quantified for the total epidermis, the basal layer, the rete ridge area and the arch area in the epidermis for the 3 LLP+ and 3 LLP- UV-exposed samples and the adjacent unexposed controls; no significant differences detected. Data are from 15 random micrographs across 2 sections per specimen ± SD.

Figure 2

Melanocyte density and epidermal thickness remains the same while keratinocyte proliferation and interdigitation increase in long-term LLP > 9 months. (A) Schematic of melanocyte-specific targets identified at the epidermal-dermal junction by immunofluorescence for TYR (tyrosinase), MITF (microphthalmia transcription factor) and MART1 (melanoma antigen recognized by T-cells). (B) Micrographs of staining for MITF, TYR and MART1 in long-term LLP+ skin and in unexposed control skin; representative micrographs are shown for #S100 (scale bar = 50 μm). (C) Quantification of melanocyte density assessed using the 3 melanocyte-specific markers quantified per basement membrane length and normalized to the respective adjacent unexposed control; data taken from 5 random micrographs across 2 sections for each marker ± SD; no significant differences detected. (D) Epidermal thickness and interdigitation index were calculated as defined in the diagram in tissue specimens using the epidermal area, epidermal length and basement membrane length as endpoints. (E) No changes in epidermal thickness occurred between LLP+ skin and adjacent unexposed controls; data taken from 5 random micrographs across 2 sections per specimen ± SEM; no significant differences detected. (F) Only the interdigitation index increased in LLP+ skin; data taken from 5 random micrographs across 2 sections per specimen ± SEM; no significant differences detected. (G) Proliferation evaluated by immunofluorescence staining for PCNA (green) in LLP+ individuals along with a melanocyte marker MART1 (red). (H) Number of PCNA-positive cells increased in LLP+ samples (P<0.05) versus unirradiated control skin, but that trend was not seen in LLP- subjects; data taken from 5 random micrographs across 2 sections for each individual.

Figure 3

Short-term LLP hyperpigmentation and heatmap of UV-induced genes identify SOX7 expression changes. (A) Image of skin pigmentation resulting from different types of UV treatments at 4 months post-exposure on the lower back of one of the subjects. Red areas are a result of previously acquired biopsies at earlier time points. (B) Microarray analysis of short-term LLP at 4 months in UV-irradiated sites compared to unexposed control skin. Heatmap clustering was generated for UVA+UVB vs the unexposed control (red (max = +3) to green (min = -3) color gradient). The orange bars at the top of heatmap indicate UV-irradiated samples and gray bars indicate the control unexposed samples. The black-dashed box highlights a cluster of known pigment genes and the blue arrow highlights SOX7. (C) Expression and distribution of SOX7 (red) and proliferating/cycling keratinocytes (stained with PCNA in green) within the rete ridge and arch areas of the epidermis corroborating the UV-induced expression changes in the heatmap (subject #17972 shown; scale bar = 50 μm).

Figure 4

Spatial disruption of hemidesmosomal components associated with SOX7. (A) Diagram of the epidermis of human skin with detailed components of hemidesmosomes found at the epidermal-dermal junction. (B) Decreases in the expression of epidermal-dermal junction components (integrin β4 and plectin) correlate with the decrease in SOX7 in long-term LLP samples >9 months after UV exposure. Representative micrographs for LLP- (#S70) and LLP+ (#S100) individuals show SOX7 (red) and the melanocyte marker MART1 (green) in the left column. Serial sections of the same subject were used to stain integrin β4 (white, middle column) and plectin (white, right column) along the basement membrane of epidermal skin sections with nuclei (blue) labeled using DAPI; scale bar = 50 μm. (C) Association between SOX7 and the hemidesmosomal component integrin β4 using proximity ligation assays in LLP+ individuals. The distribution of green puncta within the epidermis indicates an association of <40 nm between the two proteins along the epidermal-dermal junction (yellow dashed lines). scale bar = 50 μm. (D) Decreases in the total number of associations between SOX7 and integrin β4 were quantified along the basement membrane for both the rete ridge and the arch areas. At least 5 random micrographs across 2 sections were quantified for each individual. * = P<0.05, ** = P<0.005 compared to the unexposed control. No significant differences detected between LLP- areas versus unexposed control. Diagram in (A) manually created using available shapes in Microsoft PowerPoint, Redmond, WA and the add-in ScienceSlides, VisiScience, Inc., Chapel Hill, NC.

Figure 5

SOX7 knockdown indicates the potential to increases melanosome uptake by keratinocytes. (A) Western blot showing that SOX7 protein is expressed by human keratinocytes but not by human melanocytes (Lightly Pigmented: LP, Moderately Pigmented: MP, Darkly Pigmented: DP), fibroblasts, melanoma cells (A375) or breast cancer cells (T47D). (B) Workflow for SOX7 silencing and melanosome uptake assay by primary human keratinocytes. (C) Immunoblot, and corresponding Coomassie blue stained gel to show loading, indicate an effective knockdown of SOX7 as indicated by densitometry (NTC: Non-targeting control, Mel: with 1% melanosomes added). (D) Cell pellets after protein extraction show melanosome uptake following SOX7 knockdown. Tissue culture experiments were done on 3 separate occasions in duplicate, while the melanosome uptake assay was done on 2 separate occasions in duplicate. No significant differences detected.

Figure 6

Electron microscopy highlights differences in hemidesmosome density in LLP+ individuals contributing to the UV-induced hyperpigmentation model for LLP. (A) Electron micrographs of long-term LLP+ specimens (#S100) depict rete ridge areas (white lines) within the epidermis as visualized in the black box insets. The white squares in the insets indicate regions of interest shown in the larger images. Hemidesmosomes are indicated by white arrowheads along the epidermal-dermal junction (white dashed line) and are further magnified 10X in the insets at the bottom right. Large image scale bars = 0.5 μm and small black box inset image scale bars = 5 μm. (B) Hemidesmosome densities in LLP+ individuals and (C) LLP- individuals were quantified for rete ridge and arch areas of UV-treated and adjacent unexposed controls. * = P<0.05, ** = P<0.005, *** = P<0.0005 compared to the control. (D) Scheme for UV-induced hyperpigmentation for LLP development highlighting spatial modifications within the skin. Scheme manually created using available shapes in Microsoft PowerPoint, Redmond, WA and the add-in ScienceSlides, VisiScience, Inc., Chapel Hill, NC.