Wnt5a exhibits layer-specific expression in adult skin, is upregulated in psoriasis, and synergizes with type 1 interferon

Abstract

Background: Wnt5a is a member of the wingless-type patterning regulators important in pre-natal development. The expression and distribution of Wnt5a and its receptors frizzled (fzd) 3 and fzd 5 in adult human skin have not been comprehensively studied to date.

Methodology/principal findings: We here show that Wnt5a, fzd3, fzd5, as well as fzd6 are restricted to specific layers in normal epidermis, analogous to their zonal distribution in hair follicles, suggesting a role in adult skin differentiation. In line, Wnt5a and fzd5 are both overexpressed and re-distributed in the epidermis of psoriasis which involves disturbed keratinocyte differentiation. Functionally, Wnt5a lowers the concentration of IFN required to induce target genes, and increases the magnitude of IFN target gene induction, suggesting a molecular mechanism underlying IFN hypersensitivity in psoriasis. Finally, we identify nedd8 and the amyloid precursor APP, previously shown to be upregulated in psoriasis, as targets of synergistic IFNalpha/Wnt5a induction.

Conclusions/significance: The present data (i) suggest that Wnt5a regulates epidermal differentiation even in adult skin and (ii) identify synergistic induction of type 1 IFN target genes as a novel mode of Wnt5a action. Targeting Wnt5a in the skin may reduce IFN hypersensitivity and be of therapeutical value.

Figure 1. Expression of Wnt5a, Fzd3, Fzd5, and Fzd6 in adult human epidermis.

Immunohistochemistry of paraffin-embedded skin samples was performed as detailed in Methods. Panels shown are at 200× magnification.

Figure 2. Expression of Wnt5a and Fzd3, Fzd5, and Fzd6 in adult human hair follicles.

Immunohistochemistry of paraffin-embedded skin samples was performed as detailed in Methods. Panels shown are at 200× magnification. The hair follicle schematic for the cartoon on lower right was adapted from .

Figure 3. Expression of Wnt5a and Fzd proteins in psoriasis.

(a) Immunohistochemistry of Wnt5a was performed as detailed in Methods. Panels on left are 200×, panels on right are at 400× magnification. Wnt5a staining was virtually identical in n = 12 samples from independent patients.

Figure 4. Overexpression of Wnt5a in psoriatic keratinocytes in vitro.

Keratinocytes from non-affected skin from psoriasis patients or healthy control skin were expanded in vitro for 14 days, fractionated in nuclear (n) or cytoplasmic/membrane fractions (c/m), and subjected to western blot, as detailed in Methods. 50 µg of protein were loaded per lane. The bottom panel shows the Poinceau-S stain of the blot verifying even protein loading. * = non-specific band also visible in the Poinceau S stain.

Figure 5. Wnt5a induces type 1 IFN target genes and causes IFN hypersensitivity.

(a) HaCat keratinocytes were transfected with full length Wnt5a cDNA or control vector (pCDNA6.1). 12 h after transfection, the expression of nedd8, MX1, IFI-27, and APP was determined by RT-PCR, as described in Methods. The expression of Wnt5a was also determined to verify overexpression. (b) HaCat keratinocytes transfected with full-length Wnt5a cDNA (open symbols) or control vector (closed symbols) and stimulated with the indicated concentrations of IFNα for 18 h. The expression of IFI27 was then assessed using RT-PCR and quantified densitometrically. The data shown represent mean±s.d. of two independent experiments. (c) Keratinocytes from non-lesional psoriatic skin with elevated endogenous Wnt5a levels or from healthy control skin were expanded in vitro for 14 days and stimulated for 18 h with 20 ng/ml IFNα. Global gene expression was determined using the piquor skin-patho microarray. Plotted are the –fold change (IFNα vs. basal expression) of all genes (41 of 1133) exhibiting a ≥2-fold induction by IFNα. The mean fold-changes of cells from n = 3 controls are plotted along the x-axis, the mean fold-change of cells from n = 4 cells are plotted along the y-axis. Dots represent mean values, horizontal lines represent s.d. among the controls, vertical lines represent s.d. among cells from psoriasis patients. The dashed line marks the theoretical equal magnitude of gene induction.

Figure 6. Synergistic induction of APP and nedd8 by Wnt5a and IFNα.

(a) HaCat cells were transfected either with control vector or Wnt5a, followed by incubation in the absence or presence of 20 ng/ml IFNα for 18 h. Whole cell lysates were subjected to SDS-PAGE and blotted with anti-APP. The visible band migrating at appr. 70 kd is consistent with β-secretase-cleaved sAPP. Poinceau-S stain of the membrane is shown as loading control. (b) HaCat cells were transfected and stimulated with IFNα as in (a), followed by preparation of nuclear extracts, SDS-PAGE, and western blot using anti-nedd8.