Increased Expression of Wnt2 and SFRP4 in Tsk Mouse Skin: Role of Wnt Signaling in Altered Dermal Fibrillin Deposition and Systemic Sclerosis  Julie Bayle, Jennifer Fitch, Kimberly Jacobsen, Rajiv Kumar, Robert Lafyatis, Raphaël Lemaire  Journal of Investigative Dermatology  Volume 128, Issue 4, Pages 871-881 (April 2008) DOI: 10.1038/sj.jid.5701101 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Wnt mRNA expression is increased in Tsk mouse skin. (a) Representative autoradiographic images of RNase protection assays performed from skin RNA from Tsk and WT mice. Arrows point to specific mRNA of the Wnt family. (b) Graphic representation of mean relative changes in Wnt mRNA levels upon expression of Tsk-Fbn-1. Wnt levels were measured by direct β-counting and normalized to L32 expression. Data are the average of three independent experiments on 2-month-old Tsk and WT mouse littermates. Values represent the fold-increase in gene expression. Some Wnt genes may have two designations: Wnt2/Wnt2a, Wnt10b/Wnt12, Wnt13/Wnt2b. ND=not determined (expression level was low or absent). Journal of Investigative Dermatology 2008 128, 871-881DOI: (10.1038/sj.jid.5701101) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Overexpression of SFRP4 mRNA and protein in skin of Tsk mice. (a) Increased SFRP4 mRNA in skin of Tsk mice compared to control mice (WT). RNA was analyzed for SFRP4 and Fbn-1 by northern blot, quantified using phosphorimaging, and normalized to 18S ribosomal RNA. Data shown are representative of three independent experiments performed on matching pairs of 8-week-old Tsk and control mice. (b) Increased SFRP4 protein in Tsk mouse skin compared to control mouse (WT). Western blot analyses were performed using total lysate of skin from WT and Tsk mice (left panel). Expression of the 55kDa SFRP4 protein was normalized against Ponceau-stained total proteins. Protein levels were quantified by densitometry analysis. Data shown are representative of three independent experiments performed on three matching pairs of mice. Values and bars represent mean and standard error (±SEM) *P<0.05, Student's unpaired t-test, Tsk versus WT. Journal of Investigative Dermatology 2008 128, 871-881DOI: (10.1038/sj.jid.5701101) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Expression of Wnt2 and SFRP4 mRNA in various tissues of Tsk and WT mice. Total RNA was isolated from different tissues (brain, spleen, skeletal muscle, heart, skin, kidney, and liver) of 12-week-old WT and Tsk mice and analyzed for (a) SFRP4 and Fbn-1 or (b) Wnt2 by northern blot. Two SFRP4 transcripts at 1.9 and 4.2kb were detected. Results are representative of two independent experiments. Journal of Investigative Dermatology 2008 128, 871-881DOI: (10.1038/sj.jid.5701101) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Increase of SFRP4 mRNA in Tsk skin is late in development and follows increased Wnt2. RNAs from control, WT, or Tsk skin were extracted at E18.5 embryo stage and at 0, 1, 2, and 4 weeks after birth and analyzed for (a) Fbn-1 and SFRP4 or (c) Wnt2 by northern blot. mRNA levels were quantified by phosphorimaging and normalized to 18S rRNA. Values and error bars shown in (b) and (d) represent mean and standard error (±SEM). Data shown are representative of two (b) and three (d) independent experiments. Journal of Investigative Dermatology 2008 128, 871-881DOI: (10.1038/sj.jid.5701101) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Wnt3a stimulates Fbn-1 matrix formation. (a) MEFs were cultured for 10 days in the absence or presence of 100ng/ml Wnt3a. Fbn-1 and fibronectin matrix (red) were analyzed by immunofluorescence. Nuclei were stained with Hoechst (blue). Bar=10μm. The graph shows quantification of fibrils of the matrix by densitometric analysis of the fluorescent staining. (b) MEFs cultured for 16hours in the absence or presence of 100ngml−1 Wnt3a. Fbn-1 mRNA was analyzed by northern blot (left panels), and Fbn-1 and fibronectin protein levels in media by western blot (right panels). (c) Human dermal fibroblasts from normal or SSc skin were left untreated (left panel) or were treated with Wnt3a (right panel) for 10 days on chamber slides and then stained for Fbn-1 or fibronectin by immunofluorescence. Results are representative of three independent experiments performed on normal cells and SSc cells. (d) MEF-TE cells overexpressing MAGP-2 and tropoelastin-enhanced green fluorescent protein were cultured as in (a). Fbn-1 (red), MAGP-2 (red), elastic fibers (green), and fibronectin (red) were analyzed in parallel by immunofluorescence and enhanced green fluorescent protein fluorescence. Bar=10μm. (e) MEFs were serum-starved for 3hours and then treated for 1hour with TGF-β1 (5ng/ml) or Wnt3a (100ng/ml). Total lysates were analyzed by western blot for phospho-Smad3 as described in Materials and Methods. Journal of Investigative Dermatology 2008 128, 871-881DOI: (10.1038/sj.jid.5701101) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of SFRP4 mRNA is increased in the skin of SSc patients compared to healthy controls. Total RNA was extracted from the skin of four healthy control subjects (n=4) and from lesional (n=11) and non-lesional skin (n=8) areas of patients with SSc, as described in Table 2. SFRP4 expression was analyzed by quantitative real-time PCR. SFRP4 values were normalized to 18S rRNA. Horizontal lines indicate mean±SEM. Statistical significance was assessed using t-test for independent samples. *P-values less than 0.05 were considered significant: control versus lesional skin P=0.018; control versus non-lesional skin P=0.021; lesional skin versus non-lesional skin P=0.040. Journal of Investigative Dermatology 2008 128, 871-881DOI: (10.1038/sj.jid.5701101) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 SFRP4 protein is increased in the dermis of SSc patients compared to healthy controls. Punch biopsies from SSc lesional skin (n=19) and control skin (n=12), as described in Table 2, were embedded in paraffin and processed for immunohistochemistry analysis. Tissue sections of (a, c) normal healthy skin and (b, d) lesional skin were immunostained for SFRP4. Sections were counterstained with hematoxylin (blue). Arrows indicate positively stained cells (red). No staining was detected in the absence of primary antibody or with an isotype control antibody (data not shown). (a, b) Papillary dermis and (c, d) reticular dermis. Bar=20μm. (e) Graphic representation of SFRP4 staining levels in papillary and deep dermis. The staining intensity was independently quantified on each slide by three blinded investigators. Horizontal lines and error bars represent the means and ±SEM, respectively. The differences were assessed by the Mann–Whitney test. *=P<0.05, student's unpaired t-test, compared to control skin. Journal of Investigative Dermatology 2008 128, 871-881DOI: (10.1038/sj.jid.5701101) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions