c-Src activation as a potential marker of chemical-induced skin irritation using tissue-engineered skin equivalents

Abstract

Skin irritancy to topically applied chemicals is a significant problem that affects millions of people worldwide. New or modified chemical entities must be tested for potential skin irritancy by industry as part of the safety and toxicity profiling process. Many of these tests have now moved to a non-animal-based format to reduce experiments on animals. However, these tests for irritancy potential often rely on monolayer cultures of keratinocytes that are not representative of the skin architecture or tissue-engineered human skin equivalents (HSE) using complex multi-gene expression panels that are often cumbersome and not amenable for high throughput. Here, we show that human skin equivalents increase abundance of several phosphorylated kinases (c-Src, c-Jun, p53, GSK3α/β) in response to irritant chemical stimulation by phosphokinase array analysis. Specific phosphorylation of c-Src^Y419 was confirmed by immunoblotting and was plasma membrane-associated in basal/spinous cells by phospho-specific immunohistochemistry. Moreover, c-Src^Y419 phosphorylation in response to the irritants lactic acid and capsaicin was inhibited by the c-Src inhibitors KB-SRC and betaine trimethylglycine. These data provide the first evidence for c-Src specific activation in response to chemical irritants and point to the development of new modes of rapid testing by immunodetection for first-pass screening of potential irritants.

Keywords: c-Src; irritation; signal transduction; skin; toxicity.

FIGURE 1

Phosphokinase array analysis of human skin equivalents (HSE). Representative array immunoblots showing levels of phosphorylated kinases in the total protein extracts of HSE following 15 minutes treatment with H₂O as control (A), LA (B), MP (C) or Co‐DEA (D). Colour code for highlighted boxes is: , , , , , . Densitometry analysis of array immunoblots showing fold‐change abundance of phosphokinases in LA, MP or CO‐DEA treated HSE relative to water controls for Src^Y419 (E), GSK3α/β^S21/S9 (F), p53^S15 (G), p53^S46 (H) and c‐Jun^S63 (I) (n = 2 independent experiments). Immuno‐blot analysis of HSE for phospho‐c‐Src^Y419 abundance compared with total c‐Src upon stimulation with water control (con), LA, MP, Co‐DEA, CIN or CAP for 15 min (J). Densitometric analysis showing fold change in phospho‐c‐Src^{Y 419} relative to total c‐Src (K). Data are mean ± SD for n = 3 independent experiments *p < 0.05, **p < 0.01 by one‐way ANOVA with Tukey's multiple comparison post hoc test. The map accompanying the phosphokinase array is shown in Figure S2.

FIGURE 2

Abundance of phospho‐c‐Src^Y419 in tissue‐engineered human skin equivalents (HSE) in response to irritants. HSE were generated by culturing N/TERT‐1 immortalised keratinocytes on top of a human dermal fibroblast‐populated collagen scaffold before treatment with water control, LA, MP, Co‐DEA, CIN, or CAP for 15 min. Histological (haematoxylin and eosin, H&E) and immunohistochemical analysis for phospho‐c‐Src^Y419. Representative images are from three independent experiments. Scale bar = 50 μm.

FIGURE 3

c‐Src inhibitors prevent its phosphorylation in response to irritants. Immunoblots of phospho‐c‐Src^Y419 abundance compared with total c‐Src upon stimulation with water control, LA, or CAP for 15 min in the absence or presence (1‐h preincubation) of the selective c‐Src inhibitor KB‐SRC4i (A) with accompanying densitometric analysis (B) or with betaine trimethylglycine (C) with accompanying densitometric analysis (D). Representative images are from three independent experiments. Densitometry analysis for both KB‐SRC4i and betaine trimethylglycine is displayed as phospho‐c‐Src^Y419 band density relative to total c‐Src. Data are mean ± SD for n = 3 independent experiments *p < 0.05, **p < 0.01 and ***p < 0.001 by one‐way ANOVA with Tukey's multiple comparison post hoc test.