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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Fibrosis in human diseases and animal models is associated with aberrant Wnt/β-catenin pathway activation. The aim of this study was to characterize the regulation, activity, mechanism of action, and significance of Wnt/β-catenin signaling in the context of systemic sclerosis (SSc).

Methods

The expression of Wnt signaling pathway components in SSc skin biopsy specimens was analyzed. The regulation of profibrotic responses by canonical Wnt/β-catenin was examined in explanted human mesenchymal cells. Fibrotic responses were studied using proliferation, migration, and gel contraction assays. The cell fate specification of subcutaneous preadipocytes by canonical Wnt signaling was evaluated.

Results

Analysis of published genome-wide expression data revealed elevated expression of the Wnt receptor FZD2 and the Wnt target LEF1 and decreased expression of Wnt antagonists DKK2 and WIF1 in skin biopsy specimens from subsets of patients with diffuse cutaneous SSc compared to the other distinct subsets. Immunohistochemical analysis showed increased nuclear β-catenin expression in these biopsy specimens. In vitro, Wnt-3a induced β-catenin activation, stimulated fibroblast proliferation and migration, collagen gel contraction, and myofibroblast differentiation, and enhanced profibrotic gene expression. Genetic and pharmacologic approaches were used to demonstrate that these profibrotic responses involved autocrine transforming growth factor β signaling via Smads. In contrast, in explanted subcutaneous preadipocytes, Wnt-3a repressed adipogenesis and promoted myofibroblast differentiation.

Conclusion

Canonical Wnt signaling was hyperactivated in SSc skin biopsy specimens. In explanted mesenchymal cells, Wnt-3a stimulated fibrogenic responses while suppressing adipogenesis. Taken together, these results indicate that Wnts have potent profibrotic effects, and that canonical Wnt signaling plays an important role in the pathogenesis of fibrosis and lipoatrophy in SSc.

Systemic sclerosis (SSc) is a chronic autoimmune disease of unknown etiology that is associated with vascular injury, inflammatory responses, and tissue fibrosis (1). Fibrosis, the distinguishing pathologic hallmark of SSc, is characterized by overproduction of collagen and other extracellular matrix (ECM) components by fibroblasts and myofibroblasts, accompanied by progressive loss of subcutaneous adipose tissue. The source of ECM-producing activated fibroblasts within the lesional tissue of patients with SSc is controversial (2). In situ transition of fibroblasts into α-smooth muscle actin (α-SMA)–positive myofibroblasts, tissue accumulation of bone marrow–derived progenitor cells trafficking from the circulation, and transdifferentiation of epithelial cells, vascular endothelial cells, and pericytes are some of the putative mechanisms underlying the expansion of the pool of biosynthetically activated mesenchymal cells. Transforming growth factor β (TGFβ) is the master regulator of fibroblast activation and myofibroblast differentiation. Ligand binding to TGFβ receptor type I causes phosphorylation of cytoplasmic Smad2 and Smad3, promoting Smad heterocomplex formation and nuclear accumulation. The Smad complex selectively binds to Smad-binding elements, recruits the histone acetyltransferase p300 and other coactivators, and activates or represses target gene transcription (3). In addition to TGFβ, multiple cytokines and growth factors capable of inducing fibroblast activation and differentiation have been implicated in the pathogenesis of fibrosis (4).

Wnts comprise a multigene family of secreted glycoproteins that provide essential developmental signals during embryogenesis (5). Beta-catenin is a central mediator in canonical Wnt signaling (6). Binding of Wnt ligands to the cell surface receptors Frizzled and low-density lipoprotein receptor–related protein 5 (LRP-5) and LRP-6 inhibits the activation of glycogen synthetase kinase 3β (GSK-3β), which blocks β-catenin phosphorylation, ubiquitination, and degradation. Active unphosphorylated β-catenin consequently accumulates in the cytoplasm and translocates into the nucleus, where it serves as a transcriptional coactivator for the DNA binding factors lymphoid enhancer factor (LEF) and T cell factor (TCF). Although β-catenin–TCF/LEF–mediated transcription occurs ubiquitously in all tissues, the genes targeted are cell type dependent and context dependent (7). Secreted Frizzled-related proteins, Wnt inhibitory factors, and DKK proteins can interact with extracellular Wnt proteins or Wnt receptors to block Wnt/β-catenin signaling and negatively modulate Wnt responses (8). In addition, protein inhibitor of β-catenin and TCF-4 (ICAT), which is identified by yeast two-hybridization, functions as an intracellular inhibitor of Wnt/β-catenin signaling that competes for the β-catenin/TCF binding interface (9).

Whereas the importance of dysregulated Wnt/β-catenin signaling in a variety of benign and malignant human diseases has been long appreciated, its significance in the context of fibrogenesis has only recently begun to be investigated (10). Aging-associated fibrosis of muscle has been attributed to up-regulation of canonical Wnt signaling, which results in skewed progenitor cell differentiation toward a fibrogenic phenotype rather than a myogenic phenotype (11). In transgenic mice, activation of canonical Wnt signaling resulted in vigorous cutaneous wound healing and increased local collagen synthesis (12). Multiple genes involved in tissue repair and fibrosis are known to be transcriptional targets of Wnt/β-catenin, although the mechanism of their regulation is generally not well defined (13–16). Genome-wide transcriptional profiling of the lungs of patients with idiopathic pulmonary fibrosis revealed elevated expression of genes coding for Wnt ligands, receptors, regulators, and targets such as osteopontin and Wnt-1–inducible signaling pathway protein 1 (17, 18). Moreover, lung fibroblasts explanted from patients with idiopathic pulmonary fibrosis maintain activated β-catenin signaling ex vivo even in the absence of ongoing Wnt stimulus (15). Other studies showed evidence of increased Wnt expression and activity in the skin and serum of patients with SSc (19, 20). Moreover, nuclear β-catenin, a marker for active canonical Wnt signaling, was shown to be strongly up-regulated in the lungs of patients with SSc-associated pulmonary fibrosis (21). Taken together, these observations highlight a consistent association between aberrant Wnt/β-catenin signaling and pathologic fibrosis in multiple organs and species.

Because little is known about Wnt/β-catenin signaling in SSc and its relevance to disease pathogenesis, we undertook an investigation of the expression and activity of the Wnt/β-catenin axis in SSc skin biopsy specimens and in explanted human skin fibroblasts and subcutaneous progenitor cells. The results demonstrated impaired Wnt antagonism with consequent hyperactivation of canonical Wnt signaling in SSc lesional skin. Induction of Wnt signal transduction in explanted normal fibroblasts stimulated migration, proliferation, collagen gel contraction, and myofibroblast differentiation and enhanced the expression of fibrosis-related genes. In contrast, in subcutaneous adipocytes, Wnt-3a inhibited adipogenesis, at least in part, by repressing the adipogenic master regulator peroxisome proliferator–activated receptor γ (PPARγ), resulting in fibroblast differentiation with induction of type I collagen and α-SMA expression in these cells. The present findings support a key role for aberrant Wnt/β-catenin signaling in the development and progression of fibrosis in SSc and elucidate the underlying mechanism of action. Taken together with recent observations in mouse models of fibrosis and various fibrosing disorders, these results provide the rationale for exploring therapies targeting aberrant Wnt/β-catenin signaling in the treatment of SSc.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Fibroblast and subcutaneous preadipocyte cultures.

Primary cultures of neonatal fibroblasts were established by explantation from foreskins, as previously described (22). Skin biopsy specimens from healthy adult volunteers and patients with SSc were obtained following informed consent and in compliance with the Northwestern University Institutional Review Board for Human Studies (the clinical characteristics of the patients are available from the corresponding author). Embryonic fibroblasts from Smad3–/– and wild-type mice were a gift from W. H. Schnaper (Northwestern University). Unless indicated otherwise, fibroblasts were maintained at 37°C in an atmosphere of 5% CO2 in Eagle's minimum essential medium or Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% vitamins, 1% penicillin/streptomycin, and 2 mM L-glutamine (BioWhittaker). For all experiments, fibroblasts were studied between passages 4 and 8. Human subcutaneous preadipocytes from nonobese adults (Zen-Bio) were maintained in preadipocyte maintenance medium (PM-1; Zen-Bio). In selected experiments, recombinant mouse Wnt-3a (R&D Systems) or LiCl (Sigma) was added to the cultures at the indicated concentrations. The levels of active TGFβ1 in culture supernatants were determined by enzyme-linked immunosorbent assays (R&D Systems). Cell viability was determined using trypan blue dye exclusion assays.

Adipogenic differentiation.

Preadipocytes were induced to undergo adipogenic differentiation in vitro, as previously described (22). Briefly, confluent cultures were incubated in adipogenic differentiation medium (DM-2; Zen-Bio) for 4 days, followed by incubation with adipogenic maintenance medium (AM-1; Zen-Bio) for up to 7 additional days prior to harvesting. To assess intracellular lipid accumulation, cells were washed with phosphate buffered saline (PBS), fixed in 10% formalin for 60 minutes, and washed with 60% isopropanol. After drying, cells were stained with oil red O (0.5% oil red O dye in 60% isopropanol; Sigma) for 30 minutes, followed by gentle rinsing before microscopic visualization (23). Lipid-containing cells were counted in 4 random microscopic fields (400×) for each experimental condition, and experiments were repeated at least 3 times with consistent results.

RNA isolation and real-time quantitative polymerase chain reaction (qPCR).

Total RNA was isolated from confluent fibroblasts or adipocytes using TRIzol reagent (Invitrogen). Reverse transcription for real-time qPCR was performed using a SuperScript First-Strand Synthesis System (Invitrogen) according to the manufacturer's protocol. Real-time qPCR was performed using an ABI Prism 7300 Sequence Detection system (Applied Biosystems) according to the manufacturer's protocol (22). Relative messenger RNA (mRNA) expression levels were normalized to GAPDH levels in each sample and determined by calculating the ΔΔCt value, as detailed in the manufacturer's guidelines (Applied Biosystems).

Adenovirus infection, transient transfection, and RNA interference.

Fibroblasts or preadipocytes at early confluence were infected with replication-incompetent adenoviral vectors expressing human Wnt-3a (Ad-Wnt-3a) (from T. C. He, University of Chicago), constitutively active β-catenin (Ad-β-cateninca) (from J. Kitajewski, Columbia University), or green fluorescent protein (GFP; Ad-GFP). In other experiments, fibroblasts were transiently transfected with expression vectors for Frizzled-2 (OriGene) or with small interfering RNAs (siRNAs) coding for Wnt inhibitory factor 1 (WIF-1), Dkk-2, or β-catenin (CTNNB1), or scrambled control siRNAs (Dharmacon). Following incubation for the indicated periods, cultures were harvested, and whole cell lysates were subjected to Western blot analysis, or total RNA was isolated and subjected to real-time qPCR. In selected experiments, adenovirus-infected cells were transiently transfected with TOPflash reporter constructs (from R. Moon, University of Washington), which contain 8 copies of the TCF binding site upstream of luciferase, using SuperFect reagent (Qiagen). All experiments were performed in triplicate.

Cadherin-free β-catenin–binding assays.

To evaluate activation of canonical Wnt signaling, intracellular levels of cadherin-free β-catenin were determined by pull-down assays, using glutathione S-transferase (GST)/β-catenin–interacting protein (ICAT), as previously described (9). Briefly, at the end of the experiments, cells were harvested, solubilized in a nonionic detergent buffer (1% Nonidet P40 [NP40], 50 mM Tris, pH 7.5, 150 mM NaCl, and 2 mM EDTA including protease inhibitors), and centrifuged at 14,000g. Cellular β-catenin was affinity-precipitated using GST-ICAT immobilized to glutathione-coupled Sepharose (Sigma), washed with buffer A (10 mM Tris, pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.1% NP40, and 10 μg/ml leupeptin and aprotinin), and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis using β-catenin antibody (BD Biosciences).

Proliferation, migration, and gel contraction assays.

Foreskin fibroblasts infected with Ad-Wnt-3a or Ad-GFP (30 multiplicities of infection [MOI]) were seeded (1,000 cells/well) in 96-well plates. Proliferation rates were determined using CellTiter 96 Non-Radioactive Cell Proliferation Assay Kits (Promega). The modulation of cell migration was evaluated by in vitro wound-healing assays, as previously described (23). Briefly, monolayers of Ad-GFP– or Ad-Wnt-3a–infected fibroblasts were incubated in serum-free medium for 12 hours, and scratch wounds were induced using standard P1000 pipette tips. Wounds were monitored for up to 48 hours by phase-contrast microscopy, and the wound gap length was determined at 3 different sites in each sample at the indicated intervals. For collagen gel contraction assays, fibroblasts infected with Ad-GFP or Ad-Wnt-3a were seeded in type I collagen gels (BD Biosciences); following incubation in DMEM in the presence of 10% FBS for the indicated time periods, gel diameters were determined (21). The results are expressed as the percentage of gel area compared with the initial gel area.

Western blot analysis.

At the end of the incubation periods, cultures were harvested, and equal amounts of whole cell lysate proteins were subjected to electrophoresis in 4–15% Tris–glycine gradient gels (22). Proteins were then transferred to PVDF membranes, blocked with 10% fat-free milk in Tris buffered saline–Tween buffer, and incubated with the following primary antibodies: β-catenin, fatty acid binding protein 4 (FABP-4) (1:1,000; BD Biosciences), active β-catenin (1:1,000; Millipore), ICAT (1:200; Santa Cruz Biotechnology), GAPDH (1:3,000; Invitrogen), α-SMA (1:2,000; Sigma), or type I collagen (1:400; Southern Biotechnology). Membranes were washed 3 times and incubated with appropriate secondary antibodies for 45 minutes, and antigen–antibody complexes were visualized by chemiluminescence (Pierce).

Immunofluorescence analysis.

Preadipocytes were incubated in DM-2 medium for 2 days to induce adipogenic differentiation, infected with Ad-Wnt-3a (30 MOI), and incubated in AM-1 medium for an additional 96 hours. Skin fibroblasts were incubated in media with Wnt-3a for 4 hours or were infected with Ad-Wnt-3a (30 MOI) for 5 days. At the end of the incubation, cells were fixed in 4% paraformaldehyde, washed in PBS, and incubated with primary antibodies against β-catenin or α-SMA (1:1,000) for 120 minutes, followed by incubation with Alexa 488– or Alexa 594–conjugated chicken anti-mouse antibodies (Invitrogen) for 60 minutes. Nuclei were identified by DAPI staining. Nonimmune IgG was used as a negative control in each experiment. Following stringent washing, slides were examined under a Zeiss 510 UV Meta confocal microscope. Each experiment was repeated at least 3 times with consistent results.

Immunohistochemical analysis.

Immunohistochemical analysis of forearm skin biopsy specimens obtained from patients with SSc or healthy adults was performed on formalin-fixed paraffin sections, as previously described, using antibodies against β-catenin (BD Biosciences) at 1:100 dilution (24). Substitution of the primary antibody with isotype-matched irrelevant IgG served as a negative control. Nuclear β-catenin–positive and total fibroblast-like cells were counted in 4–5 random fields from 1 section/biopsy under 400× magnification.

Analysis of complementary DNA microarray data sets.

Data were analyzed from a microarray data set of genome-wide expression (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE9285), using skin biopsy specimens obtained from patients with SSc and healthy control subjects. The data set has been previously described in extensive detail (25). Genes known from the literature to be involved in Wnt signaling were extracted from the microarray data set, and those that showed expression with statistically significant differences between SSc and healthy control biopsy specimens were further analyzed. The expression level of each gene was centered on its mean value across all arrays.

Statistical analysis.

One-way analysis of variance was used to analyze the microarray data. Tukey's post hoc test was used to examine the difference between intrinsic subsets. In other experiments, the results are presented as the mean ± SD. The significance of differences between the experimental and control groups was determined by Student's t-test (using the GraphPad t-test calculator). P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Aberrant Wnt/β-catenin activation in SSc.

To gain insight into the potential significance of the Wnt/β-catenin axis in SSc, we first examined the expression of Wnt pathway components in a genome-wide expression data set that was generated using skin biopsy specimens obtained from a cohort of patients with SSc and healthy control subjects (25). This cohort comprised 17 well-characterized patients with dcSSc and 7 patients with lcSSc, along with 3 patients with localized scleroderma and 6 healthy control subjects. As shown previously, unbiased hierarchical clustering using a list of genes that showed the most consistent expression between the forearm–back pairs for each patient, but the most diversity across patients, yielded 5 intrinsic subsets distinguished by unique gene expression signatures (25). Analysis of a replication cohort that included longitudinal analysis of skin biopsy specimens has demonstrated that within an intrinsic subset, these gene expression signatures are stable (Pendergrass SA, et al: manuscript submitted).

There was no statistically significant difference between levels of the Wnt ligands (Wnt-1–Wnt-11) among the 5 intrinsic subsets. In contrast, expression of the Wnt receptor FZD2 was significantly increased (P < 0.01) in the subsets of patients with diffuse/proliferative SSc (Figure 1A). Importantly, the Wnt inhibitors DKK2 and WIF1 showed significantly decreased expression in the biopsy specimens clustering in the same intrinsic subsets composed entirely of patients with dcSSc (P < 0.01), whereas they showed increased expression in biopsy specimens clustering in the intrinsic subset of other patients with inflammatory disease (P < 0.01), comprising biopsy specimens from patients with dcSSc, patients with lcSSc, and patients with localized scleroderma.

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Figure 1. Wnt signaling is increased in systemic sclerosis (SSc). A, Analysis of Wnt pathway gene expression in skin biopsy–based microarray data sets. Microarray data sets based on skin biopsy specimens from patients with diffuse cutaneous SSc (n = 17), patients with limited cutaneous SSc (n = 7), patients with localized scleroderma (n = 3), and healthy control subjects (n = 6) were generated, and 5 intrinsic subsets were identified, as previously described (25). Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines above and below the boxes represent the maximum and minimum values, respectively. B, Immunohistochemical analysis of β-catenin. Top, Representative images of skin biopsy specimens obtained from patients with SSc and healthy control (HC) subjects. Arrows indicate nuclear β-catenin–positive fibroblast-like cells in the dermis. Original magnification × 400. Bottom, Nuclear β-catenin–positive cells in the dermis were quantified as described in Patients and Methods. Bars show the mean ± SD.

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The combination of increased Wnt receptor expression coupled with decreased expression of Wnt inhibitors in these skin biopsy specimens might be expected to give rise to hyperactivation of Wnt/β-catenin signaling. Indeed, expression of the Wnt target gene LEF1 was significantly increased in biopsy specimens clustering in the diffuse/proliferative intrinsic molecular subsets. Moreover, TCF7, a transcription factor mediating Wnt/β-catenin responses, showed similarly increased expression. As would be expected in the presence of hyperactived canonical Wnt signaling, dermal fibroblast-like cells showed increased nuclear β-catenin accumulation in SSc skin biopsy specimens (Figure 1B). Although the proportion of nuclear β-catenin–positive cells in the lesional dermis was increased in biopsy specimens from SSc patients with both early (<2 years) and late-stage disease, no significant correlation between β-catenin expression and the modified Rodnan skin thickness score was observed (data not shown).

In explanted fibroblasts, LEF1 mRNA expression was up-regulated by both Wnt-3a and infection with Ad-β-cateninca, confirming that LEF1 was a target of canonical Wnt signaling (Figure 2A). To confirm directly that elevated expression of the Wnt receptor FZD2 or reduced expression of the Wnt antagonists DKK2 or WIF1 resulted in hyperactivated Wnt/β-catenin signaling in fibroblasts, a series of gain-of-function and loss-of-function experiments were undertaken. These experiments revealed that ectopic expression of FZD2 or RNA interference–mediated knockdown of either WIF1 or DKK2 in fibroblasts enhanced their sensitivity to exogenous Wnt ligand (Figure 2B).

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Figure 2. Wnt-3a stimulates fibroblast proliferation, migration, and gel contraction. Confluent foreskin fibroblasts were infected with adenoviral vectors expressing human Wnt-3a (Ad-Wnt-3a), constitutively active β-catenin (Ad-β-Catca), or green fluorescent protein (Ad-GFP) or were transiently transfected with FZD2 or small interfering RNA (siRNA) specific for WIF1 or DKK2 or corresponding scrambled control siRNAs, followed by incubation for 24–48 hours in media with Wnt-3a (100 ng/ml) or LiCl (30 mM). A and B, RNA was isolated, and LEF1 gene expression levels were examined by real-time quantitative polymerase chain reaction. Results were normalized to GAPDH. C, Left, Whole cell lysates were subjected to glutathione S-transferase (GST)/β-catenin–interacting protein (ICAT) pull-down assays (see Patients and Methods). Active β-catenin binding to ICAT was probed with a pan-catenin antibody. Representative immunoblots are shown. Right, Fibroblasts were incubated with or without Wnt-3a for 4 hours, fixed, and probed with DAPI (blue) and β-catenin antibody (green) and examined by immunofluorescence microscopy. Representative images are shown. Original magnification × 400. D, Fibroblast migration was monitored by measuring the scratch width at 3 different sites in each sample. E, Induction of β-catenin activity by Ad-Wnt-3a treatment significantly increased fibroblast proliferation. F, Fibroblast contractility, as determined using collagen gel contraction assays, was enhanced by treatment with Ad-Wnt-3a. Values are the mean ± SD of triplicate determinations (n = 3 independent experiments). ∗ = P < 0.05 versus Ad-GFP (A, D, and F), versus time 0 (E), or versus control (B). re-Wnt-3a = recombinant Wnt-3a; PBS = phosphate buffered saline; IB = immunoblot; TGFβ = transforming growth factor β. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

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Wnt-3a–induced stimulation of profibrotic responses in normal fibroblasts.

In view of the observed hyperactivation of Wnt/β-catenin signaling in SSc, we focused on the well-characterized canonical Wnt ligand Wnt-3a. The effect of Wnt-3a on canonical β-catenin signaling was evaluated using a combination of ICAT pull-down assays, immunocytochemical analysis, and transient transfection assays. Levels of N-terminal unphosphorylated (active) β-catenin were significantly increased in Ad-Wnt-3a–infected fibroblasts as well as LiCl-treated fibroblasts (Figure 2C, left panels). Immunocytochemical analysis demonstrated that Wnt-3a induced rapid nuclear accumulation of β-catenin in these fibroblasts (Figure 2C, right panels). Transient transfection assays showed significant stimulation of the canonical Wnt reporter TOPflash by Wnt-3a infection, as well as by recombinant Wnt-3a (data not shown).

Fibroblast migration, proliferation, and collagen contraction are critical aspects of both wound healing and pathologic fibrogenesis. Modulation of wound healing by Wnt-3a was determined by in vitro scratch assays. A pipette tip was used to make a liner scratch in the confluent monolayers of fibroblasts infected with Ad-Wnt-3a, and fibroblast migration was monitored for up to 48 hours. As shown in Figure 2D, ectopic Wnt-3a signaling accelerated fibroblast migration. Moreover, inducing β-catenin activity by either recombinant Wnt-3a treatment or by Ad-Wnt-3a infection significantly increased fibroblast proliferation (Figure 2E, and data not shown). Fibroblast contractility, as determined using collagen lattice gel contraction assays, was similarly enhanced by Ad-Wnt-3a, as well as by recombinant Wnt-3a (Figure 2F, and data not shown).

To evaluate the effect of Wnt-3a on the expression of genes involved in fibrogenesis, foreskin fibroblasts were infected with Ad-Wnt-3a. Real-time qPCR showed a significant dose-dependent stimulation of COL1A2 and α-SMA mRNA expression (Figures 3A and B). The levels of both active β-catenin and total β-catenin were increased in the Ad-Wnt-3a–infected fibroblasts, as expected. Parallel experiments showed that Ad-Wnt-3a induced the formation α-SMA–positive stress fibers characteristic of myofibroblasts (Figure 3C). Consistent results were observed in multiple independent experiments with fibroblasts explanted from different donors.

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Figure 3. Wnt-3a stimulates profibrotic gene expression and myofibroblast differentiation. Confluent foreskin fibroblasts were infected with Ad-GFP or Ad-Wnt-3a (30 multiplicities of infection [MOI] or as indicated) for up to 6 days. A, RNA for COL1A2 and α-smooth muscle actin (α-SMA) was isolated at the indicated intervals and subjected to real-time quantitative polymerase chain reaction. Results were normalized to GAPDH. Bars show the mean ± SD of triplicate determinations (n = 3 independent experiments). ∗ = P < 0.05 versus Ad-GFP. B, Whole cell lysates were subjected to Western blot analysis. Representative blots are shown. C, Fibroblasts were stained with DAPI (blue) or immunostained with antibodies against α-SMA (green). Original magnification × 400. cgn = collagen (see Figure 2 for other definitions). Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

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Role of canonical TGFβ signaling in mediating Wnt-3–induced profibrotic responses.

Because TGFβ, a pivotal trigger for profibrotic responses, has been implicated in Wnt-induced myofibroblast differentiation (26), we sought to delineate its role in mediating the profibrotic effects of Wnt-3a. The results of real-time qPCR analysis showed a time- and dose-dependent increase in TGFB1 mRNA expression in fibroblasts stimulated with recombinant Wnt-3a that was prevented by siRNA-mediated knockdown of β-catenin (Figure 4A, and data not shown). Moreover, incubation with recombinant Wnt-3a, as well as with LiCl, resulted in increased Smad2 and Smad3 phosphorylation, which was prevented by preincubation of cultures with a neutralizing anti-TGFβ1 antibody, indicating the involvement of autocrine TGFβ signaling (Figure 4B). Persistent Wnt-3a expression by adenovirus infection in fibroblasts similarly induced increased levels of TGFB1 mRNA and secretion of active TGFβ1 (Figure 4C, and data not shown).

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Figure 4. Smad-dependent transforming growth factor β (TGFβ) signaling mediates profibrotic Wnt-3a responses. A–D, Confluent fibroblasts were transfected with siRNA against β-catenin (siCtnnb1) or scrambled control siRNA (A) and then incubated in media with or without Wnt-3a (100 ng/ml) for up to 24 hours (A and B) or infected with Ad-Wnt-3a for 6 days (C and D). A, TGFβ1 mRNA was isolated at the indicated intervals or following incubation with Wnt-3a for 120 minutes, and real-time quantitative polymerase chain reaction (qPCR) was performed. Results were normalized to 18S RNA. Bars show the mean ± SD of triplicate determinations in a representative experiment. B, Whole cell lysates were examined for Smad phosphorylation by Western blot analysis. Representative blots are shown. C, Levels of active TGFβ1 in culture supernatants were determined by enzyme-linked immunosorbent assay. Bars show the mean ± SD of duplicate determinations (n = 3 independent experiments). D, Cultures were incubated with or without SB431542 (10 μM) for an additional 24 hours, and COL1A2 and α-smooth muscle actin (α-SMA) mRNA expression was examined by real-time qPCR. Results were normalized to GAPDH. Bars show the mean ± SD of triplicate determinations. E, Confluent cultures of Smad3-null and wild-type mouse embryonic fibroblasts were infected with Ad-GFP or Ad-Wnt-3a. Following 6 days of incubation, Axin2 and Col1a1 mRNA were subjected to real-time qPCR. Results were normalized to the values for 36b4. Bars show the mean ± SD of triplicate determinations. ∗ = P < 0.05. See Figure 2 for other definitions.

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Complementary genetic and pharmacologic approaches were used to establish the requirement for autocrine TGFβ/Smad signaling in mediating the profibrotic effects of Wnt-3a. Pretreatment of fibroblasts with the selective activin receptor–like kinase 5 inhibitor SB431542 markedly attenuated the Wnt-3a–induced stimulation of multiple profibrotic genes (Figure 4D). Similarly, blocking TGFβ signaling with a neutralizing antibody to TGFβ abrogated the stimulation of collagen synthesis induced by Wnt-3a (data not shown). Furthermore, in Smad3-null mouse embryonic fibroblasts, Wnt-3a failed to stimulate collagen gene expression, while stimulation of Axin-2 was unaffected in these cells, indicating that Smad2/3 dependence on Wnt-3a signaling was selective for fibrogenic responses (Figure 4E). Taken together, the results from experiments using gene deletion and pharmacologic inhibition of TGFβ and Smad signaling implicate Smad2/3-mediated autocrine TGFβ signaling in the profibrotic responses induced by Wnt-3a.

Wnt-3a inhibition of preadipocyte-to-adipocyte differentiation.

The best-characterized biologic function of canonical Wnt/β-catenin signaling is cell fate specification during embryogenesis (5). In fibrosis, mesenchymal progenitor cells are a potential source of excessive myofibroblasts (27). Recent studies indicate that multipotent preadipocytes can transdifferentiate into fibroblast-like cells, suggesting that these progenitors might be another potential source of myofibroblasts (28). We have previously shown in vivo that genetic disruption of the development of adipose tissue in transgenic mice overexpressing Wnt-10b resulted in dermal fibrosis, with dense collagen deposition displacing the subcutaneous adipose layer (20).

In order to explore the possibility that Wnt-induced modulation of adipogenic differentiation might play a role in fibrogenesis, explanted subcutaneous preadipocytes were first induced to differentiate into adipocytes by incubation in DM-2 medium, followed by recombinant Wnt-3a treatment or Ad-Wnt-3a infection. The results using both approaches showed that Wnt-3a abrogated the accumulation of cytoplasmic lipid droplets induced by DM-2 (Figure 5A, and data not shown). Moreover, Ad-Wnt-3a changed the preadipocyte morphology from round to spindle-shaped with smaller nuclei (Figure 5A). The antiadipogenic effects of Wnt-3a were more pronounced in differentiating preadipocytes than in fully differentiated mature adipocytes (data not shown). Real-time qPCR showed that induction of PPARG1 and PPARG2, which are the key molecular regulators of adipogenesis, as well as fatty acid binding protein 4 (FABP-4), a well-known marker of adipocyte differentiation, was significantly suppressed in the presence of Wnt-3a (Figure 5B, and data not shown). In contrast to the profibrotic responses induced by Wnt-3a in fibroblasts, the inhibitory effects of Wnt-3a on adipogenic gene expression were Smad2/3 independent (Figure 5C).

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Figure 5. Wnt-3a suppresses preadipocyte differentiation. A, Human subcutaneous preadipocytes were incubated in preadipocyte maintenance medium (PM-1) or adipogenic differentiation medium (DM-2) for 2 days and infected with Ad-GFP or Ad-Wnt-3a (30 multiplicities of infection). After an additional 5 days, cells were fixed and stained with oil red O. Left, Wnt-3a abrogated the accumulation of cytoplasmic lipid droplets induced by DM-2. Arrowheads indicate weak red staining in differentiated adipocytes. Original magnification × 400. Right, Oil red O staining was quantified by scoring 120 cells for each condition in 5 different fields (N = none; + = small droplets, weak staining; ++ = moderate staining; and +++ = large droplets). B, Preadipocytes were incubated in media with or without Wnt-3a (100 ng/ml) for 7 days. RNA for PPARG2 and FABP4 was isolated and subjected to real-time quantitative polymerase chain reaction (qPCR). C, Preadipocytes were induced to adipogenic differentiation for 7 days. Cultures were then pretreated with SB431542 for 15 minutes and incubated with recombinant Wnt-3a (100 ng/ml) for 24 hours. RNA for PPARG1 was isolated and subjected to real-time qPCR. B and C, Results were normalized to GAPDH. Bars show the mean ± SD of triplicate determinations. ∗ = P < 0.05. See Figure 2 for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

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Wnt-3a–induced stimulation of preadipocyte differentiation into myofibroblasts.

Multipotent mesenchymal progenitor cells can be induced to differentiate into myofibroblasts (28). In order to investigate the effects of Wnt-3a on preadipocyte differentiation toward other mesenchymal cell fates, human subcutaneous preadipocytes were infected with Ad-Wnt-3a for up to 3 days. In these experiments, Wnt signaling increased the expression of α-SMA protein and mRNA in preadipocytes (Figure 6A, and data not shown). Similar results were observed with differentiated adipocytes (Figure 6A, rows 3 and 4). Furthermore, Wnt-3a stimulated type I collagen synthesis and the formation of α-SMA–positive stress fibers in these cells while simultaneously suppressing the induction of FABP-4 (Figure 6B). The induction of type I collagen by Wnt-3a was attenuated by pretreatment with the selective activin receptor–like kinase 5 inhibitor SB431542, indicating a role for TGFβ/Smad signaling in this process (Figure 6C). These results therefore demonstrate that in multipotent mesenchymal progenitor cells, Wnt-3a not only suppressed adipogenesis via negative regulation of PPARγ via autocrine TGFβ signaling but also simultaneously induced Smad2/3-dependent myofibroblast differentiation. The cumulative result of these Wnt/β-catenin–induced profibrotic and antiadipogenic activities is potent stimulation of fibroblast activation and fibrogenesis (Figure 6D).

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Figure 6. Wnt-3a stimulates preadipocytes to myofibroblast differentiation. Human subcutaneous preadipocytes were incubated in preadipocyte maintenance medium (PM-1) or adipogenic differentiation medium (DM-2) for 4 days and infected with Ad-GFP or Ad-Wnt-3a (30 multiplicities of infection) for 3 days. A, Whole cell lysates and supernatants were subjected to Western blot analysis. Representative blots are shown. B, Cells were stained with DAPI (blue) or immunostained with antibody against α-smooth muscle actin (α-SMA; red). Original magnification × 400. C, Preadipocytes incubated in DM-2 for 7 days were incubated with recombinant Wnt-3a (100 ng/ml) for 24 hours in the presence or absence of SB431542. COL1A2 mRNA was isolated and subjected to real-time quantitative polymerase chain reaction analysis. Results were normalized to GAPDH. Bars show the mean ± SD of triplicate determinations. ∗ = P < 0.05. D, Mesenchymal progenitor cells (MPCs) undergo peroxisome proliferator–activated receptor γ (PPARγ)–dependent adipogenesis or TGFβ/Smad–dependent fibrogenesis (left). In the presence of Wnt-3a (right), PPARγ is suppressed, while TGFβ is enhanced, resulting in repression of adipogenesis, myofibroblast transdifferentiation, and enhanced fibrogenesis. FABP-4 = fatty acid binding protein 4 (see Figure 2 for other definitions). Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

The Wnts have essential roles in embryonic morphogenesis, stem cell homeostasis, and cell fate determination, and genetic or acquired abnormalities of Wnt expression or signaling are associated with various diseases (29). Here, we showed that the Wnt/β-catenin pathway is hyperactivated in skin biopsy specimens from a subset of patients with dcSSc. Wnt-3a promoted myofibroblast differentiation via Smad-dependent autocrine TGFβ signaling while suppressing adipogenic differentiation of preadipocytes and inducing their differentiation into myofibroblasts. The net effect of these combined stimulatory and inhibitory activities is to promote fibrogenesis. Wnt signaling is normally tightly regulated by the balance of Wnt ligands and their inhibitors, and functional loss of Wnt antagonists results in hyperactivation of Wnt signaling (30–32). In cancers, hypermethylation of Wnt antagonists reduces their expression, with resultant increases in Wnt activity (33). Although dysregulation of Wnt signaling is implicated in oncogenesis and metastasis (34, 35), the potential relevance of Wnt signaling and its dysregulation in fibrogenesis has only recently begun to be appreciated, and the underlying mechanisms remain to be elucidated (17, 36–38).

In the present study, we observed that Wnt-3a directly induced TGFβ1 expression and activity in normal fibroblasts. Wnts and TGFβ are known to regulate the production and activity of each other in a reciprocal manner (39). Wnt-3a has been previously shown to induce expression of TGFβ and its receptors in multiple cell types in addition to fibroblasts (13, 26, 40). In silico analysis using the UCSC Genome Browser revealed enhanced transcription factor accumulation on the TGFβ1 gene promoter in β-catenin–expressing cells, suggesting direct transcriptional stimulation of TGFβ1 induced via the canonical Wnt signaling pathway (data not shown). Moreover, canonical Wnt signaling can also directly modulate the intensity of TGFβ/Smad signaling (41). Alternatively, TGFβ impacts Wnt signaling at multiple levels, including stimulation of both Wnt production and β-catenin activity (42, 43). These observations highlight the intimate reciprocal cross-regulation and extensive intracellular cross-talk that exist between the Wnt/β-catenin and TGFβ signaling pathways, which is likely to have a significant impact on fibrogenesis.

A striking finding in SSc skin is the progressive loss of subcutaneous adipose tissue and its replacement by scar tissue, resulting in characteristic skin tethering (24). Adipogenesis is under complex regulation by the nuclear hormone receptor PPARγ, which is the master regulator involved in adipogenic lineage specification (43). The Wnt/β-catenin pathway is implicated in mesenchymal stem cell fate decisions in part via suppression of PPARγ. The down-regulation of PPARγ by Wnts is thought to be mediated via a variety of mechanisms, including chicken ovalbumin upstream promoter transcription factor type II (44), microRNA (45), and epigenetic modification (46). The present results demonstrate that Wnt-3a promoted preadipocyte-to-myofibroblast differentiation while inhibiting adipogenesis in a PPARγ-dependent manner. A comparable paradigm for Wnt-regulated mesenchymal cell fate switching involving PPARγ has been reported in adipogenic/osteoblastogenic differentiation (47).

Because pathologic fibrosis in multiple human diseases and various animal models is consistently associated with aberrant Wnt/β-catenin signaling, drugs that target the Wnt cascade have enormous potential as novel therapeutic agents. Several Wnt inhibitors are in preclinical or phase I clinical trials in cancers (48). In animal models of fibrosis, inhibition of Wnt signaling by blockade of β-catenin/TCF–mediated transcription exerted potent antifibrotic effects (49, 50). Blockade of Wnt signaling with paricalcitol or the peptide mimetic ICG-001 resulted in attenuation of renal fibrosis in a mouse model (51, 52). These observations provide further support for the pivotal role of aberrant Wnt/β-catenin signaling in various forms of fibrosis and indicate the feasibility of targeting Wnts to prevent or reverse the process.

In summary, the demonstration that impaired Wnt antagonism is associated with Wnt/β-catenin pathway hyperactivation in skin biopsy specimens obtained from a subset of patients with SSc expands upon our similar findings in SSc-associated lung fibrosis (21). Canonical Wnt/β-catenin signaling in fibroblasts stimulated their proliferation, migration, gel contraction, and myofibroblast differentiation. These potent profibrotic Wnt responses involved Smad-dependent autocrine TGFβ signaling. At the same time, Wnt-3a also inhibited adipogenesis in progenitor cells and switched their differentiation toward the myofibroblast lineage. Taken together with emerging findings, these results implicate Wnt/β-catenin signaling in fibrogenesis by concomitant inhibition of adipogenesis and promotion of myofibroblast activation and differentiation. Therefore, the Wnt/β-catenin pathway is a promising target for antifibrotic therapeutic approaches.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Varga had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Wei, Fang, Lam, Gottardi, Atit, Varga.

Acquisition of data. Wei, Fang, Sargent, Hamburg, Hinchcliff, Atit.

Analysis and interpretation of data. Wei, Lam, Sargent, Gottardi, Atit, Whitfield, Varga.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We are grateful for helpful discussions with Warren Tourtellotte and members of the Varga laboratory.

REFERENCES

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  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
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