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.
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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.
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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.