Evidence for altered Wnt signaling in psoriatic skin

Abstract

The Wnt gene family encodes a set of highly conserved secreted signaling proteins that have major roles in embryogenesis and tissue homeostasis. Yet the expression of this family of important mediators in psoriasis, a disease characterized by marked changes in keratinocyte growth and differentiation, is incompletely understood. We subjected 58 paired biopsies from lesional and uninvolved psoriatic skin and 64 biopsies from normal skin to global gene expression profiling. WNT5A transcripts were upregulated fivefold in lesional skin, accompanied by increased Wnt-5a protein levels. Notably, WNT5A mRNA was markedly induced by IL-1alpha, tumor necrosis factor-alpha, IFN-gamma, and transforming growth factor-alpha in cultured keratinocytes. Frizzled 2 (FZD2) and FZD5, which encode receptors for Wnt5A, were also increased in lesional psoriatic skin. In contrast, expression of WIF1 mRNA, encoding a secreted antagonist of the Wnt proteins, was downregulated >10-fold in lesional skin, along with decreased WNT inhibitory factor (WIF)-1 immunostaining. Interestingly, pathway analysis along with reduced AXIN2 expression and lack of nuclear translocation of beta-catenin indicated a suppression of canonical Wnt signaling in lesional skin. The results of our study suggest a shift away from canonical Wnt signaling toward noncanonical pathways driven by interactions between Wnt-5a and its cognate receptors in psoriasis, accompanied by impaired homeostatic inhibition of Wnt signaling by WIF-1 and dickkopf.

Figure 1. Microarray analysis reveals that WNT5A is strikingly upregulated, and WIF1 down-regulated in the vast majority of lesional skin samples

This gene expression heat map image used transcripts from 58 paired lesional and uninvolved psoriasis and 64 normal skin samples and displays Wnt pathway members, regulators of Wnt signaling, and regulators of growth, proliferation and transcription. In addition genes involved in protein kinase activity, protein phosphatases, protein ubiquitination and other genes related to Wnt signaling are shown. Color key: red, increased expression, blue, decreased expression, as indicated in the color key and histogram (inset).

Figure 2. QRT-PCR confirmed the altered expression of several components of the Wnt signaling pathways in psoriasis

Keratin 16 (KRT16) expression was used as a positive control for lesional psoriasis skin. Data are expressed as fold-change relative to normal skin. Bars indicate mean ± S.D (n=10). Statistical significance denoted * p< 0.05, ** p<0.01, *** p<0.001.

Figure 3. Pathway analysis reveals global down-regulation of nearly all members of the canonical Wnt signaling pathway in psoriasis

Global gene expression differences between normal and lesional psoriatic skin were overlaid onto a global molecular and pathway network within the Ingenuity Pathway Knowledge Base (A). This revealed near global down-regulation (green) of nearly all members of the canonical signaling pathway. However, several genes, associated with the non-canonical Wnt pathway, were upregulated (pink) including; WNT5A and FZD5. Decreased activity of the canonical Wnt-pathway was confirmed by QRT-PCR for AXIN2, a marker of canonical Wnt signaling (B). Bars indicate mean ± S.D, * p < 0.05. Immunofluorescent microscopy of normal, uninvolved and psoriatic skin (C) (20X, n=3).

Figure 4. Immunohistochemistry of normal, uninvolved and lesional psoriasis skin revealed increased Wnt-5a and decreased WIF-1 tissue expression

Immunohistochemical staining was performed on fresh frozen sections of skin for Wnt-5a (n=5) and WIF-1 (n=5) (A). Western-blot of normal, uninvolved and lesional psoriasis n=3). Lysate from a Wnt-5a transgenic cell line was used as a positive control (B). Differences in the expression of these proteins were confirmed with computer-assisted image quantification (C). Bars indicate mean ± S.D. Magnifications 100X. Isotype-control antibodies were used and did not show any staining (not shown).

Figure 5. Expression of WNT5A by normal human keratinocytes can be induced by pro-inflammatory cytokines

WNT5A expression could be induced by 24hours' treatment with TNF-α (10ng/ml), IFN-γ (20ng/ml), IL-1α (10ng/ml) or TGF-α (24ng/ml) after 24 hours. Expression could be further induced by the additive response to IL-1α and TGF-α. Neither IL-17A (20ng/ml) nor IL-22 (20ng/ml) had any effect on WNT5A expression. Data are expressed as fold-change relative to unstimulated NHK. Bars indicate mean ± S.D (n= 3 in duplicate wells). * p<0.05, ** p<0.01.

Figure 6. Biological effects of Wnt-5a and WIF-1 on normal human keratinocytes

Conditioned medium containing Wnt-5a was obtained from a transfected cell line and diluted 1:4 in fresh M154CF culture medium. Control medium was obtained from the untransfected parental cell line. Wnt5a had growth suppressive effect on NHK that could be neutralized with anti-Wnt-5a antibodies but no effect was seen with exogenous WIF-1. Bars indicate mean ± S.D (n=3 in duplicate wells). * p<0.05, ** p<0.01 (A). CFSE staining by flow cytometry correlated with cell counts (B).