Wnt3a Induces Myofibroblast Differentiation by Upregulating TGF-β Signaling Through SMAD2 in a β-Catenin-Dependent Manner

Growing evidence suggests the Wnt family of secreted glycoproteins and their associated signaling pathways, linked to development, are recapitulated during wound repair and regeneration events. However, the role of the Wnt pathway in such settings remains unclear. In the current study, we treated mouse fibroblasts with 250 ng/mL of recombinant Wnt3a for 72 hours and examined its affect on cell morphology and function. Wnt3a induced a spindle-like morphology in fibroblasts characterized by the increased formation of stress fibres. Wnt3a decreased the proliferation of fibroblasts, but significantly increased cell migration as well as fibroblast-mediated contraction of a collagen lattice. Wnt3a significantly increased the expression of TGF-β and its associated signaling through SMAD2. Consistent with this, we observed significantly increased smooth muscle α-actin expression and incorporation of this contractile protein into stress fibres following Wnt3a treatment. Knockdown of β-catenin using siRNA reversed the Wnt3a-induced smooth muscle α-actin expression, suggesting these changes were dependent on canonical Wnt signaling through β-catenin. Neutralization of TGF-β with a blocking antibody significantly inhibited the Wnt3a-induced smooth muscle α-actin expression, indicating these changes were dependent on the increased TGF-β signaling. Collectively, this data strongly suggests Wnt3a promotes the formation of a myofibroblast-like phenotype in cultured fibroblasts, in part, by upregulating TGF-β signaling through SMAD2 in a β-catenin-dependent mechanism. As myofibroblasts are critical regulators of wound healing responses, these findings may have important implications for our understanding of normal and aberrant injury and repair events.


Introduction
Wound healing is a complex and dynamic process involving the interplay of many cellular and non-cellular components, typically culminating in the replacement of injured tissue with a fibrotic scar [1]. A number of soluble mediators released at the site of injury act as molecular cues that guide cellular responses during repair [2]. Evidence suggests the Wnt family of secreted glycoproteins and their associated signaling pathways, linked to development, are recapitulated during wound repair and regeneration events [3,4]. However, the role of Wnt signaling in this setting remains unclear.
The Wnt signaling pathway is best recognized for its critical role in development of multi-cellular organisms [5,6]. The Wnt family is comprised of 19 secreted glycoproteins that bind the Frizzled receptor and its co-receptor LRP5/6 (lipoprotein receptor-related proteins 5 or 6) to initiate an intracellular signaling cascade that controls the turnover of b-catenin (reviewed in [7]). In the absence of Wnt ligand, b-catenin is targeted for ubiquitin-mediated degradation by the 26S proteasome. Upon ligand stimulation, the canonical Wnt signaling pathway triggers a series of phosphorylation events that lead to the accumulation of cytosolic b-catenin, which then translocates to the nucleus where it binds the T-cell factor (TCF) or lymphoid enhancer binding factor (LEF) transcription factors to initiate transcription of target genes. As such, Wnt ligands can elicit a rapid and specific response in target cells.
In the current study, we examined the affect of Wnt3a, a canonical Wnt ligand, on fibroblast morphology and function. Our data suggests Wnt3a stimulates a spindle-like morphology in murine fibroblasts characterized by the increased expression of smooth muscle a-actin-positive stress fibres. These changes appear to be mediated, at least in part, by Wnt3a upregulating the expression of transforming growth factor (TGF)-b signaling through SMAD2 in a b-catenin-dependent manner. Collectively, this data suggests Wnt3a promotes the formation of a myofibroblast-like phenotype in cultured fibroblasts. As myofibroblasts are critical regulators of a wound healing response, these findings suggest a central role for Wnt signaling in regulating normal and aberrant injury and repair events.

Cell culture
Mouse embryonic fibroblasts (Clontech, product number 630914) were cultured in DMEM containing 10% FBS and 100 U/mL penicillin/streptomycin. Cells were maintained in a humidified incubator at 37uC with 5% CO 2 and used for experiments between passages 6-12. Recombinant murine Wnt3a (Peprotech, product number 315-20) was added to cells at a concentration of 250 ng/mL for 72 hours prior to performing functional studies or collecting cells for analysis. Morphological changes were photographed with a Nikon 50i series upright microscope equipped with a digital camera. Confirmation of Wnt pathway activation was performed using a TOPFlash Reporter assay, as well as qPCR gene expression of axin2, as we have previously described [8], using predesigned primers to axin2 and bactin (Applied Biosystems). In certain experiments, a TGF-b neutralizing antibody (Abcam, product number ab64715) was added during Wnt3a incubation at a concentration of 1 mg/mL. All experiments were performed in triplicate and repeated a minimum of 3 independent times.

Cell proliferation and migration assays
Cell proliferation was measured by MTS assay (Promega) 72 hours after Wnt3a treatment. Migration was measured using the in vitro scratch wound assay, as previously described [9]. Briefly, cells were treated for 72 hours with Wnt3a and confluent monolayers were scratched using a dental device to create a cellfree area where migration could be measured.

Collagen gel contraction assay
Twelve-well culture dishes were coated with 1% bovine serum albumin (BSA) and incubated for 1 hour at 37uC to create a nonstick surface that prevents gels from attaching to the dishes. Prior to performing contraction assays, fibroblasts were treated for 72 hours with Wnt3a. Cells were then trypsinized, counted and seeded into a 0.5 mg/mL Type I collagen solution (BD Biosciences, product number 354236) in growth media at a concentration of 1610 5 cells/mL. The collagen/cell suspension was vortexed, and 1 mL per well was added to the BSA-coated dishes and the solution was allowed to polymerize for 45 minutes at 37uC. Fresh growth media was added to the solidified collagen gels and plates were returned to the incubator. Collagen gel contraction was monitored over a period of 24 hours and the surface area of contracted gels was measured using Image-Pro Plus software (Media Cybernetics, Bethesda, USA).

Western blotting
Cell lysates were collected in lysis buffer (10 mM HEPES (pH 7.4), 50 mM Na 4 P2O 7 , 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 2 mM Na 3 VO 4 , and 1 mM phenylmethylsulfonyl fluoride, with 0.1% Triton X-100 and 10 mg/mL leupeptin) followed by centrifugation at high speed (14000 6 g at 4uC for 10 minutes) to recover proteins. The protein concentration of samples was measured by a Bradford protein assay. Equal amounts of protein from each sample were separated with sodium dodecylsufate-polyacrylamide gel electrophoresis (SDS-PAGE; 10% polyacrylamide) and transferred to a nitrocellulose membrane. Membranes were blocked for 1 hour in 5% milk/TBS Tween 20 and incubated overnight at 4uC with primary antibody in 2.5% milk/TBST. Following 3 washes in TBST, secondary antibody (Santa Cruz biotechnology) at a concentration of 1:2000 in 2.5% milk/TBST was added for 1 hour at room temperature. Antibody binding was visualized with the enhanced chemiluminescence detection system (Thermo Fischer Scientific). Images were captured with a Chemigenius2 system (Syngene, Frederick, USA) and band intensities were calculated with GeneTools software (Syngene).

Immunohistochemistry
Cells were fixed for 20 minutes in 3.7% formaldehyde, permeabilized with 0.1% triton X-100 for 20 minutes, blocked for 30 minutes with 1% BSA in PBS and incubated overnight at 4uC with the indicated primary antibody at a concentration of 1:100 in 1% BSA. Following primary antibody, cells were washed with PBS and incubated with anti-mouse Alexa-fluor488 conjugated secondary antibody (Invitrogen) at a concentration of 1:200 in 1% BSA for 1 hour at room temperature in the dark. To visualize f-actin, permeabilized cells were stained for 20 minutes with phalloidin conjugated to Alexa-fluor594 (Invitrogen). Cells were coverslipped with VectaShield mounting medium containing DAPI (Vector Laboratories) and images were captured using a Leica AOBS SP2 confocal microscope as we have previously described [10,11].

SiRNA transfections
Cells were seeded into 24-well culture dishes and siRNA transfection was performed using Oligofectamine (Invitrogen) as per the manufacturer's instructions. Control siRNA or mouse bcatenin siRNA (Santa Cruz Biotechnology, product numbers 37007 and 29210, respectively) were added to the cells at a concentration of 60 pmol/well for 24 hours prior to treating with Wnt3a. Cell lysates were harvested for Western blotting after 72 hours of Wnt3a treatment.

Statistical analysis
Results are represented as the mean6standard deviation. Significant differences in treatment groups were determined using the unpaired Student's t-test. For all analyses, P,0.05 was considered statistically significant.

Wnt3a induces canonical Wnt signaling in mouse fibroblasts
Mouse fibroblasts were treated for 24 hours with 250 ng/mL Wnt3a (or the vehicle control) to determine if recombinant Wnt3a induces nuclear accumulation of b-catenin. Immunohistochemistry demonstrated a strong nuclear signal for b-catenin in Wnt3atreated fibroblasts while control cells remained negative for nuclear b-catenin ( Figure 1A), suggesting Wnt3a activates canonical Wnt signaling. To confirm activation of canonical Wnt signaling by Wnt3a treatment, cells were transfected with a TOPFlash reporter construct prior to Wnt3a treatment, and a luciferase assay demonstrated that Wnt3a activated the TOPFlash reporter 5.361.6 fold after a 24 hour treatment ( Figure 1B, p,0.05). Further, mRNA expression of axin2, an early immediate target of canonical Wnt signaling, was measured after 24 hours of Wnt3a treatment. Wnt3a induced a 255671 fold increase in axin2 mRNA expression compared with vehicle treated cells ( Figure 1C, p,0.05).

Wnt3a alters the morphology of mouse fibroblasts
Wnt3a induced a marked change in fibroblast morphology after 72 hours of treatment. The Wnt-treated cells appeared spindleshaped and organized into parallel sheets as visualized by light microscopy (Figure 2A). Consistent with this, confocal microscopy showed Wnt3a-treated cells appeared larger and had altered cytoskeletons characterized by dramatically increased stress fibre formation ( Figure 2B). The increased formation of stress fibres in Wnt3a-treated fibroblasts is best visualized in low density cultures of the cells ( Figure 2C).  Wnt3a inhibited fibroblast proliferation, but increased cell migration and contraction of collagen gels Cell proliferation was observed to be significantly decreased after 72 hours Wnt3a treatment ( Figure 3A, proliferation rate: 77.464.5% of vehicle-treated cells, p,0.05). In contrast, Wnt3a significantly increased cell migration as measured by in vitro scratch wound assay ( Figure 3B, 78.162.1% vs 61.963.8%, p,0.05). Fibroblast contraction, as measured by a fibroblastpopulated collagen lattice contraction model, was also found to be significantly increased following the 72 hour Wnt3a treatment ( Figure 3C, 16.160.6% vs 29.461.3% of initial gel area, p,0.05).

Wnt3a increases TGF-b expression, SMAD2 phosphorylation and smooth muscle a-actin expression
Morphologically and functionally, our data suggests Wnt3a stimulates a myofibroblast-like phenotype in cultured fibroblasts. We next examined whether Wnt3a alters the TGF-b signaling axis in these cells. Western blot demonstrated expression of TGF-b to be upregulated after 72 hours Wnt3a treatment, and densitometry showed this change to be significant ( Figure 4A). Consistent with this, SMAD2 phosphorylation, a downstream signaling target of TGF-b, was shown to be significantly increased following Wnt3a treatment ( Figure 4B). Smooth muscle a-actin, the most commonly used marker of myofibroblast differentiation, was also found to be significantly upregulated by Wnt3a ( Figure 4C). Immunohistochemistry and confocal microscopy confirmed this, and showed the spindle-shaped fibroblasts displayed clearly visible smooth muscle a-actin-positive stress fibres following Wnt3a treatment ( Figure 4D).

Wnt3a-induced smooth muscle a-actin expression is dependent on b-catenin
To determine if the altered cell phenotype induced by Wnt3a was dependent on b-catenin, we used siRNA to knock down bcatenin expression prior to treating cells with Wnt3a. Western blot showed siRNA significantly decreased b-catenin expression in both vehicle-and Wnt3a-treated cells ( Figure 5A). Knock down of b-catenin resulted in a 48.568.4% decrease in Wnt3a-induced axin2 mRNA expression (p,0.05, data not shown), suggesting bcatenin siRNA significantly inhibited signaling through the canonical Wnt pathway. In the absence of Wnt3a, no change in SMAD2 phosphorylation was observed in cells transfected with scrambled or b-catenin siRNA. Upon stimulation with Wnt3a, however, b-catenin siRNA significantly inhibited the Wnt3ainduced SMAD2 phosphorylation ( Figure 5B). The decreased SMAD2 phosphorylation was associated with significantly decreased smooth muscle a-actin expression in Wnt3a-treated cells that had been transfected with b-catenin siRNA ( Figure 5C). No change in smooth muscle a-actin expression was observed in vehicle-treated cells. Immunohistochemistry and confocal microscopy confirmed that b-catenin knockdown inhibited the Wnt3ainduced smooth muscle a-actin expression in mouse fibroblasts ( Figure 5D).

Wnt3a-induced change in cell phenotype is dependent on TGF-b expression
To better characterize the role of TGF-b signaling in Wnt3atreated fibroblasts, a time course experiment was performed over 72 hours. Representative Western blots are shown in Figure 6A and the relative densitometry values are plotted over time in Figure 6B. Wnt3a treatment led to a rapid induction of TGF-b expression, which was highest between 12 and 24 hours after treatment. Phosphorylation of SMAD2 appeared highest between 24 and 48 hours, which was followed by the strongest expression of smooth muscle a-actin after 72 hours of treatment, indicating a sequential activation of this pathway following Wnt3a treatment.
To determine if the Wnt3a-induced SMAD2 phosphorylation and smooth muscle a-actin expression were dependent on TGF-b expression, a neutralizing antibody to TGF-b was added during Wnt3a treatment. TGF-b neutralization significantly inhibited both the Wnt3a-induced phosphorylation of SMAD2 ( Figure 6C) and smooth muscle a-actin expression ( Figure 6D). No change in SMAD2 phosphorylation or smooth muscle a-actin expression was seen in the vehicle-treated cells.

Discussion
In this study, we have shown that Wnt3a alters the phenotype of mouse fibroblasts. Structurally, Wnt3a induced a spindle-shaped morphology characterized by increased expression and incorporation of smooth muscle a-actin into stress fibres. Functionally, Wnt3a inhibited fibroblast proliferation, but increased cell migration and contraction. These changes were mediated, at least in part, by Wnt3a-induced TGF-b expression and signaling through SMAD2 in a b-catenin-dependent mechanism. Collectively, this data suggests Wnt3a stimulates the formation of a myofibroblast-like phenotype in cultured fibroblasts.
Our data are consistent with recent studies suggestive of an interaction between Wnt/b-catenin and TGF-b/SMAD signaling in controlling gene transcription and cell phenotype [12,13,14]. A recent report has demonstrated that Wnt3a controls transcriptional regulation of SM22a in mesenchymal cells via convergence with TGF-b/SMAD signaling at a novel regulatory element in the SM22a promoter [13]. SM22a is a calponin-like protein that exhibits a similar expression pattern as smooth muscle a-actin [15], both of which are smooth muscle cell contractile proteins commonly viewed as markers of an activated myofibroblast phenotype [16]. In a separate study that documented the gene expression profile induced by Wnt3a in fibroblasts, TGF-b was identified as one of the genes upregulated more than two fold after a 6 hour treatment [17]. Consistent with this, Wnt3a was found to stimulate TGF-b and collagen I mRNA expression in cultures of fetal and post-natal fibroblasts [14]. Our data add to this story by showing Wnt3a stimulates TGF-b protein expression and activation of its downstream signaling, culminating in increased smooth muscle a-actin expression. In another study, Laeremans et al showed overexpression of the Frizzled 1 receptor in combination with Wnt3a treatment stimulated the expression of myofibroblast markers in cardiac fibroblasts, changes that occurred in a b-catenin-independent pathway [18]. Interestingly, Wnt3a treatment alone was actually found to decrease the expression of smooth muscle a-actin in their study. The cause of such discrepancy is not clear at present. However, taken together, these studies support a role for Wnt3a in modifying cell phenotype, with our data strongly suggesting Wnt3a promotes a myofibroblast-like phenotype in cultured fibroblasts.
Growing evidence points to an active role for Wnt signaling in normal wound repair and in a number of human diseases. Increased canonical Wnt signaling has been observed during cutaneous wound repair [14,19,20], but is also well recognized as a contributor to a multitude of malignant disorders [21,22,23], as well as hypertrophic scarring [24], aberrant blood vessel remodeling [25], pulmonary fibrosis [26] and aging [27], among others. After tissue injury, fibroblasts differentiate into contractile and secretory myofibroblasts that participate in the synthesis and remodeling of granulation tissue during repair [28]. However, these myofibroblasts can severely impair organ function when contraction and extracellular matrix secretion become excessive [29]. Moreover, myofibroblasts present in the stroma reaction of epithelial tumors may promote the progression of cancer invasion [29,30]. TGF-b is a known and potent inducer of myofibroblast differentiation [31,32], however the regulation of TGF-b expression remains relatively understudied. The finding that Wnt3a upregulates TGF-b expression and stimulates smooth muscle aactin expression provides a link between Wnt signaling and myofibroblasts in wound repair and disease. If Wnt3a also upregulates TGF-b expression and myofibroblast differentiation in vivo, the Wnt signaling pathway may be shown as a critical regulator of the wound healing response. More work will be needed to determine how manipulating the Wnt pathway alters injury and repair events in vivo.
Wnt proteins are believed to signal through three distinct pathways, of which the canonical Wnt/b-catenin cascade is the best understood. The other pathways include the noncanonical planar cell polarity pathway and the Wnt/Ca 2+ pathway [6]. By using siRNA, we were able to demonstrate that knocking down bcatenin expression reversed the Wnt3a-induced smooth muscle aactin expression, suggesting these changes were mediated by canonical Wnt signaling through b-catenin. In addition, we demonstrated that Wnt3a also upregulated TGF-b expression. Previous reports have identified TGF-b as one of the genes whose mRNA expression is rapidly induced by Wnt3a treatment [14,17].
Thus, there appears to be sufficient evidence to suggest TGF-b is a target of canonical Wnt signaling. To our knowledge, however, there are no published reports that have examined whether TGFb gene transcription is regulated by Wnt signaling or whether its promoter contains functionally important TCF/LEF binding sites. It will be important to determine whether TGF-b is one of a growing list of direct targets genes for Wnt signaling, as this information might provide new therapeutic targets for controlling TGF-b levels in disease settings.
In summary, we provide data on a novel role for Wnt3a in stimulating myofibroblast differentiation in cultured fibroblasts. Our data suggest Wnt3a treatment promotes a contractile and migratory fibroblast phenotype that is characterized by increased expression of smooth muscle a-actin. These changes appear to be mediated by increased expression of TGF-b and signaling through SMAD2 in a b-catenin-dependent manner. As myofibroblasts play a central role in normal and aberrant injury and repair events, this data suggests Wnt3a may be critically involved in the wound healing response.