Dr. O. Distler has received consulting fees from Actelion, Pfizer, Ergonex, Bristol-Myers Squibb, Sanofi-Aventis, United BioSource, Medac, Swedish Orphan Biovitrum, Novartis, 4D Sciences, and Active Biotec (less than $10,000 each).
University of Erlangen–Nuremberg, Erlangen, Germany
Dr. J. H. W. Distler has received consulting fees, speaking fees, and/or honoraria from Actelion, Pfizer, Ergonex, Bristol-Myers Squibb, Celgene, Bayer Pharma, JB Therapeutics, Anaphore, Sanofi-Aventis, Novartis, Array Biopharma, and Active Biotec, and he owns stock in 4D Sciences.
Hedgehog signaling not only plays crucial roles during human development but also has been implicated in the pathogenesis of several diseases in adults. The aim of the present study was to investigate the role of the hedgehog pathway in fibroblast activation in systemic sclerosis (SSc).
Activation of the hedgehog pathway was analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR). The effects of sonic hedgehog (SHH) on collagen synthesis were analyzed by reporter assays, real-time PCR, and Sircol assays. Myofibroblast differentiation was assessed by quantification of α-smooth muscle actin and stress fiber staining. The role of hedgehog signaling in vivo was analyzed by adenoviral overexpression of SHH and using mice lacking 1 allele of the gene for inhibitory receptor Patched homolog 1 (Ptch+/− mice).
SHH was overexpressed and resulted in activation of hedgehog signaling in patients with SSc, with accumulation of the transcription factors Gli-1 and Gli-2 and increased transcription of hedgehog target genes. Activation of hedgehog signaling induced an activated phenotype in cultured fibroblasts, with differentiation of resting fibroblasts into myofibroblasts and increased release of collagen. Adenoviral overexpression of SHH in the skin of mice was sufficient to induce skin fibrosis. Moreover, Ptch+/− mice with increased hedgehog signaling were more sensitive to bleomycin-induced dermal fibrosis.
We demonstrated that the hedgehog pathway is activated in patients with SSc. Hedgehog signaling potently stimulates the release of collagen and myofibroblast differentiation in vitro and is sufficient to induce fibrosis in vivo. These findings identify the hedgehog cascade as a profibrotic pathway in SSc.
Systemic sclerosis (SSc) is a fibrosing connective tissue disease that primarily affects the skin but also affects the lungs, heart, and gastrointestinal tract (1). Tissue fibrosis often results in failure of the affected organs and can cause high morbidity and increased mortality. Tissue fibrosis in SSc is caused by an excessive release of extracellular matrix (ECM) by aberrantly activated fibroblasts (2). However, the molecular mechanisms underlying this pathologic and persistent activation of SSc fibroblasts are incompletely understood.
The hedgehog morphogen was first discovered in Drosophila melanogaster in 1980 (3). More than a decade later, hedgehog orthologs were identified in humans and other vertebrates (4, 5). Hedgehog ligands are highly hydrophobic secreted peptides. Three different hedgehog proteins, Sonic hedgehog (SHH), Indian hedgehog, and Desert hedgehog, have been characterized in mammals (4). SHH is the predominant hedgehog ligand in the skin (6). In the absence of hedgehog ligands, the 12-transmembrane receptor Patched homolog 1 (Ptc1) suppresses translocation of the 7-transmembrane protein Smoothened to the primary cilium and thereby prevents activation of the hedgehog pathway. However, binding of SHH to Ptc1 induces conformational changes that prevent Ptc1 from inhibiting its coreceptor Smoothened and allow accumulation of Smoothened in the primary cilium (7). Upon activation, Smoothened initiates a series of intracellular events that result in stabilization of Gli family zinc finger transcription factors, which stimulate the transcription of hedgehog target genes (8). Hedgehog signaling not only plays major roles during embryonic development (9–12), but altered activation of the hedgehog pathway has also been implicated in the pathogenesis of various diseases in adults, including a variety of different malignancies such as basal cell carcinoma, pancreatic carcinoma, and medulloblastoma (13–18).
In the present study, we aimed to characterize the role of the hedgehog pathway in fibroblast activation and tissue fibrosis in SSc. We demonstrate that overexpression of SHH results in activation of the hedgehog pathway, with accumulation of the transcription factor Gli-2 and increased expression of hedgehog target genes. SHH potently stimulated the release of collagen in cultured fibroblasts and induced the differentiation of resting fibroblasts into myofibroblasts. Moreover, overexpression of SHH in the skin of mice was sufficient to induce fibrosis, and mice lacking 1 allele of the gene for inhibitory receptor Ptc1 (Ptch+/− mice) were more sensitive to experimental fibrosis. Thus, hedgehog signaling is a novel mediator of fibroblast activation and tissue fibrosis in SSc.
PATIENTS AND METHODS
Patients and fibroblast cultures.
Biopsy specimens from patients with SSc (12 women and 2 men, median age 53 years [range 38–74 years]) were obtained from involved skin of the forearm. All patients fulfilled the criteria for SSc as described by LeRoy et al (19). Eleven patients had limited cutaneous SSc, and 3 patients had diffuse cutaneous SSc. The mean disease duration, measured from the onset of the first non-Raynaud's symptoms attributable to SSc, was 7 years (range 1–20 years). None of the patients were receiving disease-modifying antirheumatic drugs, corticosteroids, or nonsteroidal antiinflammatory drugs at the time of biopsy. Control fibroblasts (n = 12) were from skin biopsy specimens obtained from healthy age- and sex-matched volunteers. Fibroblasts from passages 4–8 were used for the experiments. All patients and control subjects signed a consent form approved by the local institutional review boards.
Immunohistochemical analysis for SHH, Gli-1, Gli-2, Gli-3, and α-smooth muscle actin (α-SMA).
Immuhohistochemical analysis for α-SMA, SHH, Smoothened, Gli-1, Gli-2, and Gli-3 was performed according to standard protocols (20). The expression of SHH, Smoothened, Gli-1, Gli-2, and Gli-3 was detected by staining with rabbit anti-human SHH polyclonal antibodies (Abcam), rabbit anti-human Smoothened polyclonal antibodies (provided by Martial Ruat), rabbit anti–Gli-1 polyclonal antibodies, rabbit anti–Gli-2 polyclonal antibodies (both from Abcam), or goat anti–Gli-3 polyclonal antibodies (Santa Cruz Biotechnology). Briefly, skin sections were deparaffinized, and epitope retrieval was performed with citrate and EDTA–Tris buffer, followed by incubation with 5% horse serum/1% bovine serum albumin for 1 hour and with 3% H2O2 for 10 minutes. Skin sections were incubated with the primary antibodies overnight at 4°C. Respective isotype antibodies were used as controls. To determine the expression of Gli-2 in fibroblasts and myofibroblasts ex vivo, skin sections were double stained with the myofibroblast marker α-SMA. Antibodies labeled with horseradish peroxidase (Dako) or alkaline phosphatase (Jackson ImmunoResearch) were used as secondary antibodies. The expression of α-SMA, SHH, and Gli-2 was visualized with diaminobenzidine peroxidase substrate solution (Sigma-Aldrich), and, for double-staining, the expression of α-SMA was visualized with BCIP/nitroblue tetrazolium (Vector).
Stimulation with SHH.
Dermal fibroblasts were stimulated with recombinant SHH (R&D Systems) dissolved in 0.1% bovine serum albumin/phosphate buffered saline in concentrations from 0.3 μg/ml to 1.0 μg/ml for 24 hours. Stimulation with recombinant transforming growth factor β (TGFβ) at a concentration of 10 ng/ml dissolved in 4 mM HCl containing 1% bovine serum albumin served as a positive control.
Total RNA was isolated with a NucleoSpin RNA II extraction system (Machery-Nagel). Reverse transcription into complementary DNA (cDNA) was performed using random hexamers (21, 22). Gene expression was quantified by TaqMan and by SYBR Green real-time PCR using an ABI Prism 7300 Sequence Detection System (Applied Biosystems). The following primer pairs were used for analyses: for human GLI2, forward 5′-CTGCTCGAAGGCCTACTCC-3′, reverse 5′-ACCGCAGGTGTGTCTTCAG-3′; for human ACTA2, forward 5′-GAACATGCCATCATCACCAA-3′, reverse 5′-TGGTGCCAGATCTTTTCCAT-3′; for human proα1(I), forward 5′-ACGAAGACATCCCACCAATC-3′, reverse 5′-ATGGTACCTGAGGCCGTTC-3′; for human proα2(I), forward 5′-GGTCAGCACCACCGATGTC-3′, reverse 5′-CACGCCTGCCCTTCCTT-3′; for human PTCH1, forward 5′-ACAAACTCCTGGTGCAAACC-3′, reverse 5′-GCTGATGTCGATGGGCTTAT-3′; for human PTCH2, forward 5′-TGTGGTGGGAGGCTATCTG-3′, reverse 5′-GCATGGTCACACAGGCATAG-3′; for human cyclin D1, forward 5′-AACCTGAGGAGCCCCAAC-3′, reverse 5′-AAGCGTGTGAGGCGGTAG-3′. Samples without enzyme in the reverse transcription were used as negative controls. Nonspecific signals caused by primer dimers were excluded using dissociation curve analysis and samples without cDNA. A predeveloped β-actin assay (Applied Biosystems) was used to normalize for the amounts of loaded cDNA. Differences were calculated using the threshold cycle method, and the comparative threshold cycle method was used for relative quantification.
Total soluble collagen in cell culture supernatants was quantified using a Sircol collagen assay (Biocolor). The total collagen content of the skin was determined using hydroxyproline assays, as previously described (23).
Messenger RNA (mRNA) stability assay.
To investigate whether SHH stabilizes collagen or fibronectin mRNA, an mRNA stability assay was performed as previously described (24). Healthy fibroblasts were stimulated with 1 μg/ml of SHH. Actinomycin D at a concentration of 10 μg/ml was added 6 hours later. Unstimulated fibroblasts with or without actinomycin D served as controls. Fibroblasts were harvested 0, 4, 8, 12, 18, and 24 hours after the addition of actinomycin D, and levels of collagen mRNA were analyzed by real-time PCR.
Luciferase reporter assay.
To analyze whether SHH activates transcription of the COL1A2 gene, a luciferase reporter assay was performed (25). The −53 COL1A2-Luc construct containing the fragment between −353 and +58 nucleotides of the proα1(II) gene was kindly provided by M. Trojanowska (26). HEK 293T cells were seeded into 6-well plates 1 day before transfection. Reporter construct (0.5 μg) and polyethylenimine were mixed and added to the cells in serum-free medium. A common LacZ reporter vector was used as control. Cells were stimulated with SHH directly after transfection. Luciferase activity was analyzed 24 hours after transfection.
Immunofluorescence for α-SMA.
Healthy dermal fibroblasts were seeded into chamber slides and stimulated with recombinant SHH for 24 hours. Afterward, fibroblasts were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.25% Triton X-100. Blocking experiments were performed with 5% horse serum followed by incubation with monoclonal anti–α-SMA antibodies (clone 1A4; Sigma-Aldrich) at 4°C overnight. Secondary antibodies labeled with tetramethylrhodamine (Invitrogen) were used for visualization. Nuclei of fibroblasts were counterstained with DAPI. The amount of fluorescence was quantified using ImageJ 1.43u software (National Institutes of Health).
Stress fiber staining.
To analyze whether SHH induces development of stress fibers, healthy dermal fibroblasts were stimulated with SHH for 24 hours. Afterward, fibroblasts were fixed with 4% PFA and permeabilized with 0.25% Triton X-100. After blocking with 1% bovine serum albumin, stress fibers were visualized with phalloidin (27). The amount of fibers with fluorescent staining was quantified using ImageJ 1.43u software.
Adenoviral overexpression of SHH.
DBA/2 mice were purchased from Charles River. The adenoviral expression constructs encoding for SHH (AdSHH) and control viruses (adenovirus-expressing green fluorescent protein [AdGFP]) have previously been described (28). One group of mice received injections with AdSHH, whereas another group injected with AdGFP served as a control group (n = 6 each). On days 0 and 28, each mouse received an injection of 6.6 × 107 plaque-forming units into a defined area (1 cm2) on the upper back. All mice were killed on day 56.
Bleomycin-induced dermal fibrosis in Ptch+/− mice.
A potential worsening of experimental fibrosis in Ptch+/− mice (29) was evaluated in a mouse model of bleomycin-induced dermal fibrosis (25, 30). Four groups of mice were analyzed: 2 groups consisted of Ptch+/− mice on a 129/Pas background, whereas the other 2 groups consisted of wild-type littermates (Ptch+/+). One group received a subcutaneous injection of 100 μl bleomycin every other day into a 1-cm2 section of the upper back, whereas the other 2 groups received subcutaneous injections of 0.9% NaCl for 3 weeks. Six mice were analyzed in each group.
After the mice were killed, the injected skin sections were removed, fixed in 4% formalin, and embedded in paraffin. Histologic sections were stained with hematoxylin and eosin for the determination of dermal thickness. Dermal thickness at the injection sites was analyzed with a Nikon Eclipse 80i microscope at 100× magnification. Dermal thickness was determined by measuring the largest distance between the epidermal–dermal junction and the dermal–subcutaneous fat junction, as previously described (31). The analysis was performed by an experienced examiner who was blinded to the treatment of the mice.
Activation of TGFβ and canonical Wnt signaling in the skin of mice.
The experimental procedure for induction of fibrosis by intradermal injection of mice with replication-deficient adenovirus type 5 overexpressing a constitutively active TGFβ receptor type I construct has previously been described (32). The generation and the phenotype of mice expressing a conditional allele of a stabilized mutant of β-catenin, the central integrator of canonical Wnt signaling, selectively in fibroblasts under a tamoxifen-inducible Col1a2 promoter (β-catenin [Δexon3] × Col1a2-CreER mice) have also been described previously (33).
Data are expressed as the mean ± SEM. Wilcoxon's signed rank tests for related samples and the Mann-Whitney U test for nonrelated samples were used for statistical analyses. P values less than 0.05 were considered significant.
Activation of hedgehog signaling in SSc.
To investigate whether the hedgehog pathway is activated in dermal fibrosis, we first analyzed the expression of the ligand SHH. Weak staining for SHH was detectable in normal healthy skin (Figure 1A). In contrast, prominent overexpression of SHH was detected in fibroblasts, endothelial cells, and keratinocytes of the fibrotic skin of patients with SSc (Figure 1A).
To confirm that the overexpression of SHH results in increased activation of the hedgehog pathway in SSc, we analyzed expression of the hedgehog transcription factors Gli-1, Gli-2, and Gli-3 (Figure 1B). In particular, the expression of GLI2 was increased in the skin of patients with SSc compared with healthy controls. The expression of GLI1 was also enhanced in patients with SSc, although staining was less intense than that for GLI2 (additional information is available from the corresponding author). Consistent with the data obtained by immunohistochemical analysis, the mRNA levels of GLI1 and GLI2 were increased in skin biopsy specimens obtained from patients with SSc compared with controls, with a mean ± SEM fold increase of 6.4 ± 1.1 for GLI1 mRNA and an increase of 3.6 ± 0.3 for GLI2 mRNA. The expression of GLI3 was detectable only in scattered cells in patients with SSc and was virtually absent in healthy individuals (additional information is available from the corresponding author). Double-staining for α-SMA and GLI1 or GLI2 demonstrated a prominent accumulation of both GLI transcription factors in myofibroblasts (results not shown). Seventy percent of myofibroblasts were positive for GLI1, and 82% were positive for GLI2.
We also quantified the mRNA levels of PTCH1, PTCH2, and cyclin D1, which are classic downstream target genes of hedgehog signaling (34). We observed significantly elevated mRNA levels of PTCH1, PTCH2, and cyclin D1 in the skin of patients with SSc compared with healthy controls (Figures 1C–E). Taken together, these data demonstrated increased activation of the hedgehog pathway in the skin of patients with SSc.
Induction of SHH by TGFβ and canonical Wnt signaling.
TGFβ signaling and canonical Wnt signaling are key pathways in the pathogenesis of SSc. Interestingly, both pathways have also been identified as regulators of hedgehog signaling (35, 36). To analyze whether activation of TGFβ or canonical Wnt signaling might stimulate the expression of SHH and thereby activate hedgehog signaling in vivo, we analyzed the mRNA levels of SHH in skin samples from mice infected with replication-deficient adenovirus type 5 overexpressing a constitutively active TGFβ receptor type I and mice expressing a conditional allele of a stabilized mutant of β-catenin, the central integrator of canonical Wnt signaling, selectively in fibroblasts under a tamoxifen-inducible Col1a2 promoter (β-catenin [Δexon3] × Col1a2-CreER mice).
Potent activation of TGFβ signaling was confirmed by the demonstration of nuclear translocation of phosphorylated Smad3 and increased transcription of TGFβ target genes such as Pai1 and Ctgf. Activation of canonical Wnt signaling was confirmed by the demonstration of nuclear β-catenin in fibroblasts and increased transcription of the target gene Axin2. Activation of both TGFβ and canonical Wnt signaling strongly increased the mRNA levels of SHH in the skin of mice, suggesting that the up-regulation of TGFβ and canonical Wnt signaling in SSc might drive the activation of hedgehog signaling in fibrotic murine skin (Figure 2A). Consistent with these in vivo findings, increased levels of SHH were also observed in fibroblasts stimulated with recombinant TGFβ and recombinant Wnt-1 (data not shown). Moreover, we confirmed that stimulation of cultured fibroblasts with TGFβ in the absence of exogenous SHH is sufficient to induce the transcription of the hedgehog target genes Ptch1, Ptch2, and CycD (Figure 2B).
SHH stimulates the release of ECM and induces fibrosis.
After demonstrating that the hedgehog pathway is activated in the skin of patients with SSc, we further investigated the effects of pathway activation in cultured fibroblasts. Although a trend toward increased levels of Gli-1 and Gli-2 in SSc fibroblasts was observed, this did not reach statistical significance. Stimulation with recombinant SHH significantly increased the mRNA levels of COL1A1 and COL1A2 (Figures 3A and B). Consistent with higher mRNA levels, we also observed increased levels of collagen protein in the cell culture supernatant (Figure 3C). Of note, the stimulatory effects of SHH were comparable with those observed with TGFβ, which is considered to be the most potent profibrotic mediator. The stimulatory effects of recombinant SHH on the expression of type I collagen were comparable between SSc fibroblasts and fibroblasts from healthy individuals.
SHH-induced release of ECM is mediated by enhanced transcription of COL1A1 and COL1A2.
To determine the mechanism by which SHH increases mRNA levels of type I collagen, mRNA stability and reporter assays were performed. The relative decreases in the mRNA levels of COL1A1 and COL1A2 upon incubation with actinomycin D over time were not altered by SHH. However, SHH increased the transcription of type I collagen and stimulated the expression of luciferase under control of the COL1A2 promoter in a dose-dependent manner, by up to a mean ± SEM of 98 ± 31% (P < 0.05) (Figures 4A and B), suggesting that SHH stimulates the transcription of type I collagen without increasing mRNA stability. We analyzed the promoters of COL1A1 and COL1A2 for Gli binding sites with the consensus sequence GACCACCCA. We did not observe GACCACCCA consensus sequences within 2 kbp upstream of COL1A1 and COL1A2. Although these findings do not exclude the possibility of a role for nonconserved binding sites or very-far-upstream binding sites, the results may provide evidence either for Gli-independent effects or for indirect effects of Gli transcription factors with up-regulation of other transcription factors that directly bind to and activate the transcription of type I collagen.
SHH induces myofibroblast differentiation.
Based on the observations that hedgehog signaling was particularly active in myofibroblasts, and that myofibroblasts were key effector cells in fibrotic diseases, we further investigated the role of SHH in myofibroblast differentiation. Myofibroblasts can be identified by the expression of α-SMA and the formation of stress fibers. Stimulation with SHH increased the protein levels of α-SMA in resting dermal fibroblasts in a dose-dependent manner, to a mean ± SEM of 807 ± 327% (P = 0.007) (Figures 5A and B). Consistent with the induction of α-SMA, SHH induced the formation of stress fibers in resting fibroblasts (Figures 5C and D). In the absence of SHH, only weak phalloidin staining was observed in scattered cells. However, upon stimulation with SHH, intensive phalloidin staining with thick stress fiber bundles were detected in all fibroblasts (Figure 5D), and the mean ± SEM amount of fluorescence was increased to 3,475 ± 961% (P < 0.001) (Figure 5C). The stimulatory effects of SHH on myofibroblast differentiation were comparable between SSc fibroblasts and fibroblasts from healthy individuals. Again, the stimulatory effects of SHH on the expression of α-SMA and on stress fiber formation were comparable with those of TGFβ. These findings demonstrated that SHH potently induced differentiation of resting fibroblasts into metabolically active myofibroblasts.
Overexpression of SHH induces dermal fibrosis in mice.
After demonstrating that pathway activation induced an activated, SSc-like phenotype in healthy fibroblasts in vitro, we next analyzed whether overexpression of SHH is sufficient to induce skin fibrosis in vivo. The adenovirus-mediated overexpression of SHH and subsequent activation of the hedgehog pathway in AdSHH-injected mice was confirmed by immunohistochemistry for Gli-2 (additional information is available from the corresponding author). An increased accumulation of ECM was observed upon subcutaneous injection of AdSHH (Figure 6A). Dermal thickness was significantly increased, by a mean ± SEM of 52 ± 11%, after AdSHH treatment compared with AdGFP treatment (P = 0.01) (Figure 6B). Consistent with increased dermal thickness, the myofibroblast counts and the hydroxyproline content were significantly higher after AdSHH treatment than after AdGFP treatment (Figures 6C and D). These results demonstrated that overexpression of SHH is sufficient to induce skin fibrosis in vivo.
Increased sensitivity of Ptch+/− mice to bleomycin-induced skin fibrosis.
To confirm by an additional approach that enhanced hedgehog signaling results in increased sensitivity to fibrosis, we analyzed experimental fibrosis in Ptch+/− mice (29). The lack of 1 allele of Ptch1 results in increased activation of the hedgehog pathway upon ligand binding due to decreased inhibition of Smoothened by Ptch1 (37). Although heterozygosity of PTCH does not occur in SSc, these mice mimicked the enhanced hedgehog signaling observed in patients with SSc. Local injection of bleomycin induced the accumulation of collagen in the dermis of Ptch+/+ mice and Ptch+/− mice. However, dermal fibrosis was more pronounced in Ptch+/− mice than in Ptch+/+ mice (additional information is available from the corresponding author). Also, the increase in dermal thickening and the myofibroblast counts upon bleomycin challenge were significantly higher in Ptch+/− mice than in Ptch+/+ mice (additional information is available from the corresponding author). Thus, the lack of 1 allele of the inhibitory receptor Ptch1 caused higher sensitivity to experimental fibrosis in vivo.
Here, we demonstrated that hedgehog signaling activates fibroblasts in vitro and in vivo and plays a prominent role in tissue fibrosis in SSc. Different factors might drive the overexpression of SHH and the subsequent activation of the hedgehog pathway in SSc. Hypoxia has been reported to induce the expression of SHH and activate the hedgehog pathway. Increased expression of SHH was observed in neuronal progenitor cells and neurons upon exposure to an oxygen concentration similar to that observed in the skin of patients with SSc (38). We previously demonstrated severely reduced oxygen tension in the skin of patients with SSc, with overexpression of several oxygen-regulated genes (39, 40). Thus, hypoxia might contribute to activation of the hedgehog cascade. In addition, the overexpression of SHH might be driven by profibrotic cytokines such as platelet-derived growth factor (PDGF) and TGFβ or by Wnt proteins, all of which are overexpressed in fibrotic diseases. PDGF stimulated the expression of SHH in hepatic stellate cells in vitro and in keratinocytes in vivo (41, 42). TGFβ promoted survival of immature liver cells and caused surviving mature hepatocytes to release SHH (43). Indeed, this study is the first to present evidence that TGFβ and Wnt signaling increases the mRNA levels of SHH in cultured fibroblasts and in murine skin, suggesting a hierarchical system in which TGFβ signaling and Wnt signaling stimulate hedgehog signaling. However, further experiments are needed to confirm this hypothesis.
SHH stimulated resting fibroblasts to differentiate into myofibroblasts and potently enhanced the release of collagen. Of note, the potency of the stimulatory effects of SHH was comparable to that of TGFβ, which is currently considered the most potent stimulus for fibroblast activation. Overexpression of SHH in mice resulted in accumulation of collagen and dermal thickening, demonstrating that SHH alone is sufficient to induce fibrosis in vivo. The crucial role of hedgehog signaling was further highlighted by the finding that mice lacking 1 functional allele of the inhibitory receptor Ptch1, which results in increased activation of the hedgehog pathway upon ligand binding due to decreased inhibition of Smoothened by Ptch1 (37), are more sensitive to experimental fibrosis. The prominent role of hedgehog signaling in fibrosis is underlined by recent studies in mouse models of experimental bile duct ligation and nonalcoholic fatty liver disease (44). The importance of the hedgehog pathway for tissue fibrosis in humans is also supported by characteristic changes in the stroma surrounding tumors, with activated hedgehog signaling. Pancreatic tumors and medulloblastomas are commonly characterized by a desmoplastic reaction, with formation of an excessive ECM capsule around the tumors (45). Thus, hedgehog signaling might play a central role in the pathogenesis of fibrosis, and inhibition of the hedgehog pathway might be a promising strategy for the treatment of fibrotic disorders.
In summary, we demonstrated that the hedgehog pathway is activated in SSc and plays a central role in the pathophysiology of the disease. SHH induces myofibroblast differentiation, stimulates the release of collagen with potency similar to that of TGFβ, and also is sufficient to induce fibrosis in vivo. These findings identify hedgehog signaling as a novel pathway contributing to the aberrant activation of SSc fibroblasts.
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. J. H. W. Distler 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. Dees, Akhmetshina, Gusinde, Roudaut, Traiffort, Ruat, O. Distler, J. H. W. Distler.
Acquisition of data. Horn, Palumbo, Dees, Tomcik, Zerr, Avouac, Gusinde, Roudaut, Traiffort, Ruat, J. H. W. Distler.
Analysis and interpretation of data. Horn, Palumbo, Cordazzo, Akhmetshina, Tomcik, Gusinde, Zerr, Zwerina, Roudaut, O. Distler, Schett, J. H. W. Distler.