Interaction of Wnt5a with Notch1 is Critical for the Pathogenesis of Psoriasis

Abstract

Background: Psoriasis is characterized by uncontrolled hyperproliferation, aberrant differentiation, and dermal infiltration of immune cells. Recent studies have reported that Wnt5a and Notch1 signaling are altered in psoriatic skin lesions.

Objective: We aimed to investigate the interaction of Wnt5a with Notch 1 with respect to inflammation-mediated epidermal hyperproliferation in psoriasis.

Methods: Expression of Wnt5a and Notch1 signaling-related proteins were examined in psoriatic skin biopsies. Wnt5a was upregulated in human keratinocytes by treating the cells with its recombinant form (rWnt5a).

Results: In psoriatic lesions, expression of Wnt5a increased while that of Notch1 decreased when compared to that in non-lesional and normal skin. Treatment with rWnt5a increased the proliferation of keratinocytes and increased their secretion of interleukin (IL)-23, IL-12, and tumor necrosis factor (TNF)-α. Further, exposure of keratinocytes to IL-1α, TNF-α, transforming growth factor-α, and interferon-γ downregulated Notch1 as well as HES 1, which is downstream to Notch1, but increased the Wnt5a levels. The upregulated Wnt5a in keratinocytes downregulated both Notch1 and HES1.

Conclusion: Our data suggest that Wnt5a and Notch1 signaling exert counteracting influences on each other and are involved, in part, in the pathomechanism of psoriasis.

Keywords: Notch1; Psoriasis; Wnt5a.

Fig. 1. Wnt5a expression in psoriasis. (A) Quantitative reverse transcription polymerase chain reaction results show that Wnt5a is upregulated approximately 2.4-fold in psoriatic lesional skin compared with that in non-lesional samples (n=10, paired samples). The box plot represents the log ratio of the expression levels in lesional and non-lesional psoriatic skin samples. Upper and lower borders of the boxes indicate 75th and 25th percentiles, respectively, whereas horizontal lines within boxes denote medians. (B) Immunohistochemical analyses show that Wnt5a is increased in all the layers of psoriatic lesions (left) compared with non-lesional skin (right). Samples were stained with DAB (3,3'-diaminobenzidine; brown color) and counterstained with hematoxylin (purple).

Fig. 2. Notch1 and hairy and enhancer of split (HES) 1 expression in psoriasis. (A) Quantitative reverse transcription polymerase chain reaction results did not yield any difference in the mRNA levels of NOTCH1 between psoriatic lesional and non-lesional skin samples, but revealed a decrease in HES1 mRNA expression in the former. (B) Immunohistochemical analyses (DAB [3,3'-diaminobenzidine] staining, ×400) showed that protein levels of Notch1 (upper panel) were decreased in all the layers of psoriatic lesions (left) compared with non-lesional skin samples (right). HES1 (lower panel) was also decreased at the protein level in psoriatic lesional samples.

Fig. 3. The effects of Wnt5a on normal human keratinocyte (NHK) proliferation. NHKs were treated with (A) rWnt5a (100 ng/ml) and (B) siRNAs against Wnt5a for 2 and 3 d, and then examined by Cell Proliferation Bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay. The graph shows the percentage of BrdU-positive cells with respect to the control. Data represent the mean±standard deviation. Con: control, d: day. ^*p-value<0.05, determined by the Student's t-test.

Fig. 4. Migration assays in neonatal normal human keratinocytes (NHKs) and human dermal fibroblasts (HDFs). NHKs (A) and HDFs (B) were cultured in either Wnt5a-conditioned or control media for 3 d, following which the cells (A: 1×10⁶, B: 5×10⁴) were incubated on transwell membranes (8-µm pore size) for 24 h. Migrated cells were stained with hematoxylin and eosin, and then enumerated under a microscope (×100).

Fig. 5. Association of Wnt5a and Notch1 expression with the cytokine environment. (A) Neonatal normal human keratinocytes (NHKs) were stimulated with tumor necrosis factor (TNF)-α, interferon (IFN)-γ, transforming growth factor (TGF)-α, and interleukin (IL)-1α, either alone or with a cytokine mixture containing TNF-α, IFN-γ, TGF-α, and IL-1α for 24 h. Western blot analyses detected the expression of Notch1, hairy and enhancer of split (HES) 1, and Wnt5a. (1) Untreated NHKs (control), (2) IL-1α (10 ng/ml), (3) TNF-α (10 ng/ml), (4) TGF-α (24 ng/ml), (5) IFN-γ (20 ng/ml), (6) cytokine mixture of IL-1α (10 ng/ml), and TNF-α (10 ng/ml), (7) IL-1α (10 ng/ml), TNF-α (10 ng/ml), TGF-α (24 ng/ml), and IFN-γ (20 ng/ml)-treated NHKs. (B) Analysis of rWnt5a-treated NHK culture media by enzyme-linked immunosorbent assay (ELISA). Cells were treated with rWnt5a (100 ng/ml) for 6, 24, 48, and 72 h, following which the culture media were harvested. TNF-α and IL-1α production (pg/ml) were determined by ELISA. Data represent the mean±standard deviation. ^*p-value<0.05, ^**p-value<0.01; determined by the Student's t-test. (C) NHKs were treated with rWnt5a (100 ng/ml) for 1, 3, 6, 24, and 72 h, following which the expression levels of NOTCH1 and HES1 were determined by reverse transcription polymerase chain reaction.

Fig. 6. Phospho (p)-AKT signaling in rWnt5a-treated neonatal normal human keratinocytes (NHKs). NHKs, serum-deprived for 24 h, were either treated with 100 ng/ml of rWnt5a or left untreated, and harvested after 15 min, 30 min, 1 h, and 3 h. Proteins in the cell lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by western blotting using antibodies against p-AKT and total AKT. (A) Representative western blot for total AKT and p-AKT, showing an increase in the phosphorylated (activated) form of AKT in rWnt5a-treated NHKs. (B) Intensities of p-AKT and total AKT signals were determined using the BioRad VersaDoc Imaging System. The p-AKT/total AKT ratio is indicated for each time point.