The EGFR-ERK/JNK-CCL20 Pathway in Scratched Keratinocytes May Underpin Koebnerization in Psoriasis Patients

Abstract

Epidermal keratinocytes represent a rich source of C-C motif chemokine 20 (CCL20) and recruit CCR6⁺ interleukin (IL)-17A-producing T cells that are known to be pathogenic for psoriasis. A previous study revealed that scratch injury on keratinocytes upregulates CCL20 production, which is implicated in the Koebner phenomenon characteristically seen in psoriasis patients. However, the molecular mechanisms leading to scratch-induced CCL20 production remain elusive. In this study, we demonstrate that scratch injury upregulates the phosphorylation of epidermal growth factor receptor (EGFR) and that the specific EGFR inhibitor PD153035 attenuates scratch-induced CCL20 upregulation in an extracellular signal-related kinase (ERK)-dependent, and to a lesser extent, a c-Jun N-terminal kinase (JNK)-dependent but p38 mitogen-activated protein kinase (MAPK)-independent manner. Immunoreactive CCL20 was visualized in the keratinocytes that lined the scratched wound. IL-17A also induced the phosphorylation of EGFR and further augmented scratch-induced CCL20 upregulation. The EGFR-ERK/JNK-CCL20 pathway in scratched keratinocytes may explain why Koebnerization is frequently seen in psoriasis patients.

Keywords: CCL20; ERK; IL-17A; JNK; Koebner phenomenon; epidermal growth factor receptor; keratinocyte; p38 MAPK; psoriasis; scratch injury.

Figure 1

Scratch injury–induced CCL20 production. The production of CCL20 was measured at 3, 6, and 24 h after the initiation of culture in non-scratched control and scratched keratinocyte cultures. Representative data of three independent experiments are shown. ** p < 0.01. *** p < 0.001.

Figure 2

Scratch injury-induced CCL20 production depends on activation of epidermal growth factor receptor (EGFR). (A) Scratch injury-induced CCL20 production was measured in the presence or absence of PD153035 (EGFR inhibitor, 300 or 600 nM) at 24 h after scratching. (B) The phosphorylation of EGFR (P-EGFR) was measured by western blot analysis at 1 h after scratching. Representative enzyme-linked immunosorbent assay (ELISA) data and Western blot images of three independent experiments are shown. ** p < 0.01. *** p < 0.001.

Figure 3

Immunofluorescent visualization of CCL20 at 6 h after scratching. (A) Non-scratched control keratinocytes stained with control IgG. (B) Non-scratched control keratinocytes stained with anti–CCL20 antibody. (C) Scratched keratinocytes stained with control IgG. (D) Scratched keratinocytes stained with anti–CCL20 antibody. Original magnification 100×. Representative data of three independent experiments are shown.

Figure 4

Scratch injury induces phosphorylation of ERK1/2, JNK, and p38 MAPK at 1 h after scratching. Phosphorylation of ERK1/2, JNK, and p38 MAPK was examined by western blotting in non-scratched control and scratched keratinocytes. Representative blot images of three independent experiments (A) and the relative expressions of phosphorylated proteins (B) are shown. * p < 0.05. ** p < 0.01. *** p < 0.001.

Figure 5

Scratch-induced CCL20 (24 h after scratching) upregulation depends on ERK1/2 and JNK activation. The CCL20 production was measured in nonscratched control and scratched keratinocytes in the presence or absence of U0126 (ERK1/2 inhibitor, 10 μM), SB203580 (p38 MAPK inhibitor, 10 μM), and SP600125 (JNK inhibitor, 10 μM). Representative data of three independent experiments are shown. * p < 0.05. ** p < 0.01. *** p < 0.001.

Figure 6

IL-17A augments scratch-induced CCL20 upregulation at 3 h after scratching. (A) CCL20 production was measured in non-scratched control and scratched keratinocytes in the presence or absence of IL-17A (0.1 or 1 ng/mL). (B) Non-scratched keratinocytes were stimulated with IL-17A (0.1 or 1 ng/mL) with or without PD153035 (EGFR inhibitor, 300 nM). Representative data of three independent experiments are shown. * p < 0.05. ** p < 0.01. *** p < 0.001.

Figure 7

IL-17A upregulates the phosphorylation of EGFR at 3 h after scratching. The phosphorylation of EGFR was measured by western blot in non-scratched control keratinocytes and scratched-keratinocytes in the presence or absence of IL-17A (1 ng/mL) or PD153035 (EGFR inhibitor, 300 nM). Representative blot images of three independent experiments and the relative expressions of phosphorylated proteins are shown. * p < 0.05. ** p < 0.01. *** p < 0.001.