Role of ERK Pathway in the Pathogenesis of Atopic Dermatitis and Its Potential as a Therapeutic Target

Abstract

Atopic dermatitis (AD) is an eczematous skin disorder characterized by type 2 inflammation, barrier disruption, and intense itch. In addition to type 2 cytokines, many other cytokines, such as interferon gamma (IFN-γ), interleukin 17 (IL-17), and interleukin 22 (IL-22), play roles in the pathogenesis of AD. It has been reported that the extracellular signal-regulated kinase (ERK) is downstream of such cytokines. However, the involvement of the ERK pathway in the pathogenesis of AD has not yet been investigated. We examined the expression of p-ERK in mouse and human AD skin. We also investigated the effects of the topical application of an ERK inhibitor on the dermatitis score, transepidermal water loss (TEWL), histological change, and expression of filaggrin, using an AD-like NC/Nga murine model. The effects of an ERK inhibitor on filaggrin expression in normal human epidermal keratinocytes (NHEKs) and on chemokine production from bone marrow-derived dendritic cells (BMDCs) were also evaluated. p-ERK was highly expressed in mouse and human AD skin. Topical application of an ERK inhibitor alleviated the clinical symptoms, histological changes, TEWL, and decrease in expression of filaggrin in the AD-like NC/Nga murine model. The ERK inhibitor also restored the IL-4 induced reduction in the expression of filaggrin in NHEK, and inhibited chemokine production from BMDC induced by IL-4. These results indicate that the ERK pathway is involved in the pathogenesis of AD, and suggest that the ERK pathway has potential as a therapeutic target for AD in the future.

Keywords: ERK pathway; TEWL; atopic dermatitis; barrier function; filaggrin.

Figure 1

The expression of p-ERK in mouse and human skin. Immunohistochemical staining for p-ERK in normal (A,D) and AD (B,E) skin. Scale bar: 100 μm (C) Percentage of intraepidermal p-ERK expression of control (n = 2) and AD (n = 8) in mice. (F) Percentage of intraepidermal p-ERK expression of control (n = 6) and AD (n = 17) in human. All data are presented as mean ± standard error of the mean (S.E.M.). *** p < 0.001 Student’s unpaired two-tailed t-test.

Figure 2

Topical application of ERK inhibitor alleviates the clinical symptoms of mite antigen-induced AD in NC/Nga mice. (A) Schema of induction of mite antigen-induced AD-like murine model. After 2 h treated with 4% SDS, mite antigen, Dermatophagoides farinae extract (Biostir AD^®), was epicutaneously applied to the upper back and ear skin of mice twice a week for three weeks to develop AD. These mice were then topically treated with ERK inhibitor or vehicle for 7 days on the same site. (B) Macroscopic features of skin lesions of control, AD, and AD + U0126 groups on day 0 and 6. (C,D) Total dermatitis score (C) and all dermatitis scores such as erythema, dryness, edema, and excoriation (D) were assessed on day 0, 2, 4, and 6. All data are presented as mean ± S.E.M. (n = 9 per group). * p < 0.05, ** p < 0.01, *** p < 0.001 Student’s unpaired two-tailed t-test on each day.

Figure 3

Topical application of ERK inhibitor improves the histological changes of mite antigen-induced AD in mice. Histological appearance (hematoxylin-eosin staining) of skin lesions of control (A), AD (B), and AD + U0126 (C) groups on day 7. Scale bar: 50 μm. (D) Number of total inflammatory cells in dermis on the dorsal skin of mice. Measurements of epidermal thickness and epidermal + dermal thickness ** p < 0.01, *** p < 0.001 one-way analysis of variance followed by Bonferroni’s multiple comparison test. (E) Immunohistochemical staining for intraepidermal p-ERK expression of skin lesions of AD and AD + U0126 groups. Scale bar: 100 μm., *** p < 0.001 Student’s unpaired two-tailed t-test on each day. All data are presented as mean ± S.E.M. (n = 3 control group, n = 8 AD group, n = 8 AD + U0126 group).

Figure 4

Topical application of ERK inhibitor improved TEWL and restored FLG expression in mite antigen-induced AD in mice. (A) TEWL was measured on the lesion by a Vapo Scan instrument on day 0, 2, 4, and 6. Data were presented as the average of five points repeated recordings. All data are presented as mean ± S.E.M. (n = 9 per group). *** p < 0.001 Student’s unpaired two-tailed t-test on each day. Immunohistochemical analysis of FLG protein (green) in the back skin in control (B), AD (C), AD + U0126 (D) groups on day 7. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm.

Figure 5

ERK inhibitor restored the reduction in the expression levels of filaggrin and involucrin in NHEK. NHEKs were treated with IL-4 (20 ng/mL) in the presence or absence of ERK inhibitor (1 µM) for 72 h (FLG) or 48 h (IVL) for qRT-PCR and for 96 h (FLG) or 72 h (IVL) for Western blotting analyses. FLG (A) and IVL (B) mRNA expression was analyzed by qRT-PCR. FLG (A) and IVL (B) protein expression was analyzed by Western blotting. All data are presented as mean ± S.E.M. (n = 3 per each group). ** p < 0.005, *** p < 0.001 one-way analysis of variance followed by Bonferroni’s multiple comparison test.

Figure 6

ERK inhibitor inhibited chemokine production from BMDC induced by IL-4. BMDCs were treated with IL-4 (20 ng/mL) in the presence or absence of ERK inhibitor. (A,B) CCL17 and CCL22 mRNA expression was analyzed by qRT-PCR. (C,D) CCL17 and CCL22 protein expression was analyzed by ELISA. All data are presented as mean ± S.E.M. (n = 3 per each group). * p < 0.05, ** p < 0.005, *** p < 0.001 one-way analysis of variance followed by Bonferroni’s multiple comparison test.